Alternative ingredients in practical diets for Pacific white shrimp (Litopenaeus vannamei)

by

Xuan Qiu

A dissertation submitted to the Graduate Faculty of Auburn University

in partial fulfillment of the requirements for the Degree of

Doctor of Philosophy

Auburn, Alabama May 6, 2017

Keywords: Apparent digestibility coefficients, Bacterial biomass, Fermented yeast, Growth response, Novel soybean cultivars, Ulva meal

Copyright 2017 by Xuan Qiu

Approved by

D. Allen Davis, Chair, Professor of Aquatic Animal Nutrition Claude E. Boyd, Professor of Water Quality

Terry Hanson, Professor of Aquaculture Economics Asheber Abebe, Professor of Mathematics and Statistics

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ABSTRACT

It is a well-known that fish meal supplies will not increase as most fisheries are beyond

sustainable limits. If aquaculture and shrimp production in particular is expected to expand, the

industry must move away from fish meal as a primary protein source, particularly in the

production diets. As shrimp are a major aquaculture product and a primary use of fish meal it is

critical that we expand our knowledge of novel protein source which could be used to reduce fish

meal levels in production diets. Towards this goal the present study was dedicated to explore the

potential of novel ingredients (flash dried yeast, non-genetically modified soy cultivars, bacterial

biomass, fish meal analogue, and Ulva meal) as protein sources in practical diets for Pacific

white shrimp, L. vannamei.

The first study was design to evaluate the potential of a novel yeast product flash dried

yeast produced by low pH fermentation of Saccharomyces Pombe as a feed supplement in

practical shrimp feed. Under the conditions of this study, the energy and protein digestibility of

flash dried yeast are significantly lower than FM and SBM. Amino acids digestibility of FDY

was lowest among the three ingredients tested. The use of FDY at 60 g kg-1 caused significant

negative impacts on growth, feed conversion ratio and protein retention. Dietary flash dried yeast

supplementation in the practical diets for Pacific white shrimp had no effects on the proximate

composition of the whole shrimp body. Based on these results, further research regarding the

effects of the low levels (< 40 g kg-1) inclusion of FDY in practical diets on immune responses of

Pacific white shrimp is warranted.

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The second study explored non-genetically modified soy cultivars as protein sources in

commercial type shrimp feed formulations. The results indicate that breeding technology and

novel soy processing has the potential to increase the nutritional values of SBM for shrimp feeds.

Observed trends on immune indicators of shrimp to both independent and combined effects of

soy ingredients and fermented yeast were not easily discernible. The variable response may be

related to the difficult in working with shrimp or a suboptimal exposure period.

The third study investigated the effects of a dried fermented biomass as alternative

ingredients for fish meal or soy protein concentrate. Under the reported conditions of the study,

the use of dried fermented biomass can partially replace fish meal up to 50 g kg-1 without

causing negative effects on the growth performance of Pacific white shrimp. However,

completely replacement of fish meal (100 g kg-1) by dried fermented biomass resulted in growth

depression. These results were confirmed in a second trial, which replaced soy protein

concentrate with fermented biomass dried under two methods. The granular dried fermented

biomass worked well as a substitution for soy protein concentrate, however, the inclusion of

spray dried dry fermented biomass at 60 and 120 g kg-1 decreased the growth of shrimp. This

data demonstrates that granular dried fermented biomass is a good nutrient sources and can be

incorporated in practical shrimp feed formulations.

The aim of the fifth study was to evaluate a novel bacterial biomass as a replacement for

soybean meal in practical shrimp feeds. Under the conditions of the present study bacterial

biomass can be utilized up to 4% in shrimp feed without causing growth depression. However,

supplementations (≥ 6%) of bacterial biomass can result in negative effects on growth response,

FCR, and protein as well as amino acids retention efficiency. Negative result of dry matter

digestibility as well as no improvements in the treatments balanced on digestible protein basis

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infers that something other than protein is influencing performance. Given that this is a new

technology, there is a need to evaluate BB in term of possible immune stimulation as well as a

nutrient source.

The sixth study was designed to evaluate a fish meal analogue as a replacement for fish

meal in practical shrimp feed. Results indicated that in a practical diet containing 20% fish meal,

fish meal analogue can replace all of the fish meal as long as the diets are supplemented with

inorganic phosphorus without compromising the growth of shrimp. The improvement of growth

when fish meal analogue was incorporated at 4.95% across three trials was not able to be

defined. Given the good growth across the range of inclusion without any indication of a growth

depression, the digestibility of the protein of fish meal analogue would be similar to that of the

fish meal for which it was substituted. Hence, the low nutrient digestibility of fish meal analogue

may due to an atypical response or the product simply does not work with the testing technique.

The seventh study evaluated the potential of Ulva meal Ulva sp. as an alternative protein

source to fish meal and soybean meal in practical shrimp feed. Results demonstrated a clear

depressing in growth as fishmeal was replaced. This data also demonstrated significant

difference between batches of Ulva with the second batch producing the poorest results. To

elucidate if digestible protein was limiting growth, a trial was initiated for which feeds were

formulated on a digestible protein basis. In this trial, growth and survival were significantly

reduced as the level of Ulva meal (Batch 2) was increased. Although, growth and survival was

depressed this was less than that of previous trials, indicating that protein quality may be part of

the problem. However, given the level of protein replacement other components of Ulva meal are

likely to be causing poor performance. To survey possible problems caused by high levels of

minerals the meals and select diets were analyzed for mineral content. Clearly there are shifts in

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mineral profiles; however, there is no obvious correlation to a mineral and this research team

feels that it is unlikely a mineral toxicity. Other possible reasons which are beyond the scope of

this project but would include anti-nutrients present in the algae. If Ulva meals are to be use to

their full potential, e.g. as a primary protein source, the anti-nutritional components will need to

be identified, specific lines of plants with enhanced nutrient value need to be developed and of

course processing technologies evaluated to produce a high quality commercial product.

There is a clear need to develop sustainable ingredient sources for aquaculture, the use of

soybean meal is clearly a viable option. Besides soybean meal, there are abundant alternative

sustainable ingredients such as yeast, bacterial biomass, seaweed meals, and etc, which exhibited

great potential as both immune enhancer and protein source. Therefore, it is vital for us to

evaluate these potential alternative ingredients and promote a sustainable, environmentally

friendly, and economical aquaculture.

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ACKNOWLEDGEMENTS

This dissertation is dedicated to my major advisor Dr. Donald A. Davis. First, I would

like to appreciate the opportunity that he provided for me to participate in his alternative

ingredients for Pacific white shrimp nutrition program. Without his patience, encouragement,

and support, I would hardly complete this thesis. Also, I am grateful to all my other committee

members, Dr. Claude E. Boyd, Dr. Hanson, and Dr. Ash Abebe for their participation and

suggestions in completing this program. Specially thanks to my parents for their infinite love,

encouragement, and support. Also, I would like to show my thanks to all the members in Dr.

Donald A. Davis nutrition lab Guillaume Salze, Melanie Rhodes, Sirirat Chatvijikul, Lay

Nguyen, May Myat Noe Lwin, Charles Roe, Mingming Duan, Ha Van To Pham Thi, Jingping

Guo, Hongyan Tian, Alexandra Amorocho, Elkin Montecino Perez, Romi Novriadi. They helped

and supported me a lot. I offer my deep appreciation to friends, colleagues, students, professors,

and staffs of Swingle Hall for their friendship, hospitality, assistance, knowledge, and support

during the completion of this project. Last but not least, many thanks to Archer Daniels Midland

Company, Binational Agricultural Research & Development Fund (BARD), Hatch Funding

Program of Alabama Agriculture Experiment Station, H.J. Baker & Brothers Inc., Knipbio Inc.,

Navita Premium Feed Ingredients Inc., for providing funding for this research.

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Table of Contents

ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... vi LIST OF TABLES ....................................................................................................................... xiii LIST OF FIFURES........................................................................................................................xx CHAPTER I .................................................................................................................................... 1 Introduction ..................................................................................................................................... 1 REFERENCES ............................................................................................................................... 6 CHAPTER II ................................................................................................................................. 11 EVALUATION OF FLASH DRIED YEAST AS A NUTRITIONAL SUPPLEMENT IN PLANT BASED PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei....................................................................................................................................................... 11 1. Introduction ............................................................................................................................... 11 2. Materials and Methods .............................................................................................................. 13

2.1 Experimental diets .......................................................................................................... 13 2.2 Growth trial ..................................................................................................................... 13 2.3 Digestibility trial ............................................................................................................. 15 2.4 Statistical analysis ........................................................................................................... 16

3. Results ....................................................................................................................................... 16 4. Discussion ................................................................................................................................. 18 5. Conclusion................................................................................................................................ 22

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6. References................................................................................................................................. 22 CHAPTER III ............................................................................................................................... 37 EVALUATION OF THREE NON-GENETICALLY MODIFIED SOYBEAN CULTIVARS AS INGREDIENTS AND A YEAST-BASED ADDITIVE AS A SUPPLEMENT IN PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei ............................................... 37 1. Introduction ............................................................................................................................... 37 2. Materials and Methods .............................................................................................................. 39

2.1 Ingredients preparation ................................................................................................... 39 2.2 Experiment design and diets ........................................................................................... 39 2.3 Growth trials ................................................................................................................... 40 2.4 Physiological assessment ................................................................................................ 41 2.5 Water quality monitoring ................................................................................................ 43 2.6 Statistical analysis ........................................................................................................... 44

3. Results ....................................................................................................................................... 44

3.1. Water quality .................................................................................................................. 44 3.2. Growth trials .................................................................................................................. 44 3.3. Physiology trial .............................................................................................................. 46

4. Discussion ................................................................................................................................. 47 5. Conclusion ................................................................................................................................ 52 References ..................................................................................................................................... 53 CHAPTER IV ............................................................................................................................... 70 EVALUATION OF DRIED FERMENTED BIOMASS AS A FEED INGREDIENT IN PLANT-BASED PRACTICAL DIETS FOR JUVENILE PACIFIC WHITE SHRIMP Litopenaeus vannamei ....................................................................................................................................... 70 1. Introduction ............................................................................................................................... 70 2. Material and Methods ............................................................................................................... 71

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2.1. Experimental design and diets ....................................................................................... 71 2.2. Growth trials .................................................................................................................. 72 2.3. Water quality monitoring ............................................................................................... 73 2.4. Statistical analysis .......................................................................................................... 73

3. Results ....................................................................................................................................... 74

3.1. Water quality .................................................................................................................. 74 3.2. Growth trials .................................................................................................................. 74 3.3. Whole body composition ............................................................................................... 75 3.4. ANCOVA output ........................................................................................................... 75

4. Discussion ................................................................................................................................. 75 5. Conclusion ................................................................................................................................ 81 References ..................................................................................................................................... 81 CHAPTER V ................................................................................................................................ 95 EVALUATION OF A NOVEL BACTERIAL BIOMASS AS A SUBSTITUTION FOR SOYBEAN MEAL IN PLANT-BASED PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei .................................................................................................................. 95 1. Introduction ............................................................................................................................... 95 2. Material and Methods ............................................................................................................... 96

2.1. Experimental design and diets ....................................................................................... 96 2.2. Growth trials .................................................................................................................. 98 2.3. Water quality monitoring ............................................................................................. 100 2.4. Digestibility trial .......................................................................................................... 100

2.5. Statistical analysis ........................................................................................................ 101

3. Results ..................................................................................................................................... 102

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3.1. Water quality ................................................................................................................ 102 3.2. Growth trials ................................................................................................................ 102 3.3. Whole body composition ............................................................................................. 103 3.4. Protein and amino acid retentions ................................................................................ 104 3.5. Digestibility trial .......................................................................................................... 105

4. Discussion ............................................................................................................................... 105 5. Conclusion .............................................................................................................................. 110 References ................................................................................................................................... 110 CHAPTER VI ............................................................................................................................. 133 EVALUATION OF A FISH MEAL ANALOGUE AS A REPLACEMENT FOR FISH MEAL IN PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei ................. 133 1. Introduction ............................................................................................................................. 133 2. Materials and Methods ............................................................................................................ 134

2.1. Experimental design and diets ..................................................................................... 134 2.2. Growth trials ................................................................................................................ 135 2.3. Water quality monitoring ............................................................................................. 137 2.4. Digestibility trial .......................................................................................................... 137 2.5. Statistical analysis ........................................................................................................ 139

3. Results ..................................................................................................................................... 139

3.1. Water quality ................................................................................................................ 139 3.2. Growth trial .................................................................................................................. 140 3.3. Digestibility trial .......................................................................................................... 141

4. Discussion ............................................................................................................................... 142

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5. Conclusion .............................................................................................................................. 147 References ................................................................................................................................... 147 CHAPTER VII ............................................................................................................................ 168 UTILIZATION OF GREEN SEAWEED ULVA sp. AS A PROTEIN SOURCE IN PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei ............................................. 168 1. Introduction ............................................................................................................................. 168 2. Materials and Methods ............................................................................................................ 170

2.1. Experimental diets ....................................................................................................... 170 2.2. Growth trials ................................................................................................................ 172 2.3. Water quality monitoring ............................................................................................. 174 2.4. Digestibility trial .......................................................................................................... 174 2.5. Statistical analysis ........................................................................................................ 176

3. Results ..................................................................................................................................... 176

3.1. Water quality ................................................................................................................ 176 3.2. Growth performances ................................................................................................... 177 3.3. Proximate composition and amino acid profile of whole shrimp body ....................... 178 3.4. Protein and amino acids retention ................................................................................ 179 3.5. Regression analysis ...................................................................................................... 181 3.6. Digestibility trial .......................................................................................................... 181

4. Discussion ............................................................................................................................... 182

4.1. Ingredient composition ................................................................................................ 182 4.2. Nutrient digestibility .................................................................................................... 184 4.3. Growth performance and survival ............................................................................... 186 4.4. Body compositions ...................................................................................................... 191

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4.5. Protein and amino acid retention ................................................................................. 192

5. Discussion ............................................................................................................................... 194 References ................................................................................................................................... 194

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LIST OF TABLES Chapter II Table 1 Formulation and chemical composition of test diets used in the growth trial. ................ 29 Table 2 Proximate composition, phosphorus content, and amino acid profile of the ingredients

used in the growth and digestibility trials. ............................................................................ 30 Table 3 Proximate composition (g kg-1 as is) and amino acid profile (% as is) of the test diets

used in the growth trial. ........................................................................................................ 31 Table 4 Composition of reference diet for the determination of digestibility coefficients of flash

dried yeast (FDY), fishmeal (FM), and soybean meal (SBM). ............................................ 32 Table 5 Performance of juvenile Pacific white shrimp L. vannamei (1.78±0.03g) offered diets

with different flash dried yeast (FDY) levels (0, 10, 20, 40, and 60 g kg-1) for six weeks. . 33 Table 6 Proximate analysis of whole shrimp body offered diets with different flash dried yeast

(FDY) levels (0, 10, 20, 40, and 60 g kg-1) for six weeks. .................................................... 34 Table 7 Apparent dry matter (ADMD), apparent energy (ADE) and apparent protein (ADP)

digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. ....................................................................... 35

Table 8 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal

(FM) and flash dried yeast (FDY) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. .................................................................................................... 36

Chapter III Table 1 Formulation and chemical composition of test diets used in the growth trial ................. 57 Table 2 Proximate analysis (as is g 100 g-1), trypsin inhibitors levels (TIU g-1), acid detergent

fiber (ADF) and neutral detergent fiber (NDF) (as is g 100 g-1), phosphorus (as is g 100 g-1), urease activity (pH increase), protein dispersibility index (PDI), starch (as is g 100 g-1) and amino acid (AA) profiles (as is g 100 g-1) of solvent extracted soybean meal (SBM), navita 1, 2, and 3 utilized in the trials. ............................................................................................. 59

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Table 3 Proximate analysis (as is g 100 g-1), trypsin inhibitors levels (TIU/g), and amino acid (AA) profiles (as is g 100 g-1) of the test diets utilized in the trials. ..................................... 61

Table 4 Response of juvenile Litopenaeus vannamei (Initial weight 0.15±0.01g, 46 days in trial

1; initial weight 0.20±0.01g, 35 days in trial 2) offered diets with different soybean ingredients at varying levels and w/o fermented yeast. ........................................................ 63

Table 5 Principle component analysis of trypsin inhibitors and essential amino acids of diets. .. 65 Table 6 Multiple linear regression of weight gain (WG) and feed conversion ratio (FCR) with

first three principle components (PC1 PC2 and PC3). ......................................................... 66 Table 7 Pearson correlation coefficients of growth performances (final biomass, final mean

weight, and weight gain), feed conversion ratio (FCR), and survival with trypsin inhibitors, methionine and protein levels of the diets. The first line of each cell is the value of correlation coefficient and the second line of each cell is P-value. ...................................... 67

Table 8 Two-way analysis output of stress and immune response of Pacific white shrimp L.

vannamei fed with different soy sources and fermented yeast product levels. ..................... 68 Chapter IV Table 1 Composition (g kg-1 as is) of test diets utilized in trial 1. ................................................ 86 Table 2 Composition (g kg-1 as is) of test diets utilized in trial 2. ................................................ 87 Table 3 Proximate composition, and amino acid profile of dried fermented biomass used in trial

1 (DFB) and 2 (SDFB and GDFB have identical nutrient compositions), fish meal (FM), soy protein concentrate (SPC), and corn protein concentrate (CPC). ................................... 89

Table 4 Proximate composition and amino acid profile of the test diets used in trial 2. .............. 90 Table 5 Performance of juvenile shrimp L. vannamei (Initial weight 0.59 g) offered diets with

different dried fermented biomass (DFB) levels (0, 25, 50, and 100 g kg-1) for six weeks in trial 1. .................................................................................................................................... 92

Table 6 Performance of juvenile shrimp L. vannamei (Initial weight 2.34 g) when shrimp were

offered diets with different levels (0, 20, 40, 60, and 120 g kg-1) of spray dry (S) and granular (G) dried fermented biomass (DFB) for six weeks in trial 2. ................................. 93

Table 7 Proximate analysis (g kg-1) of whole shrimp body when shrimp were offered diets with

different levels (0, 20, 40, 60, and 120 g kg-1) of spray dry (S) and granular (G) dried fermented biomass (DFB) for six weeks in trial 2. ............................................................... 94

Table 8 Analysis of covariance (ANCOVA) output of performance and proximate composition

of whole shrimp body data in trial 2. .................................................................................... 95

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Chapter V Table 1 Composition (% as is) of test diets utilized in trial 1. .................................................... 116 Table 2 Composition (% as is) of test diets utilized in trial 2. .................................................... 117 Table 3 Composition (% as is) of test diets utilized in trial 3. .................................................... 118 Table 4 Composition of reference diet for the determination of digestibility coefficients of

bacteria biomass (BB) ......................................................................................................... 119 Table 5 Proximate composition (% as is), phosphorus content (% as is), and amino acid profile (%

as is) of the ingredients used in the growth and digestibility trials. .................................... 120 Table 6 Proximate composition (% as is) and amino acid profile (% as is) of the test diets used in

trial 1. .................................................................................................................................. 121 Table 7 Proximate composition (% as is) and amino acid profile (% as is) of the test diets used in

trial 2. .................................................................................................................................. 122 Table 8 Proximate composition1 (% as is) and mineral composition1 (g kg-1: phosphorus, sulfur,

potassium, magnesium, calcium, sodium; mg kg-1: iron, manganese, copper, zinc) of the test diets used in trial 3. ............................................................................................................. 123

Table 9 Performance of juvenile shrimp L. vannamei (Initial weight 1.51g) offered diets with

different bacterial biomass levels (0, 6, and 12 %) for six weeks in trial 1. ....................... 124 Table 10 Performance of juvenile shrimp L. vannamei (Initial weight 0.98g) offered diets with

different bacteria biomass levels (0, 1, 2, 4, 6, and 12 %) for six weeks in trial 2. ............ 125 Table 11 Performance of juvenile shrimp L. vannamei (Initial weight 0.15g) offered diets

formulated to partially replace soybean meal on a digestible protein basis for six weeks in trial 3. .................................................................................................................................. 126

Table 12 Proximate composition2 (moisture: % as is; protein and lipid: % dry weight) and amino

acid profile2 (% dry weight) of whole shrimp body offered diets with different bacteria biomass levels (0, 1, 2, 4, 6, and 12 %) for six weeks in trial 2. ........................................ 127

Table 13 Protein2 and amino acids3 retention efficiencies of Pacific white shrimp at the

conclusion of a 6-week growth trial 2 in which shrimp were offered diets formulated to contain different bacteria biomass levels (0, 1, 2, 4, 6, and 12 %). .................................... 129

Table 14 Proximate composition of whole shrimp body and protein retention efficiency (PRE)

offered diets formulated to utilize bacterial biomass (BB) partially replace soybean meal on a digestible protein basis for six weeks in trial 3. ............................................................... 131

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Table 15 Apparent dry matter (ADM), apparent energy (AED) and apparent protein (APD)

digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. ..................................................................... 132

Table 16 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal

(FM), bacteria biomass (BB) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. ............................................................................................................ 133

Chapter VI Table 1 Composition (% as is) of test diets used in the growth trial 1 and 2. ............................ 154 Table 2 Composition (% as is) of test diets used in the growth trial 3. ...................................... 155 Table 3 Composition of reference diet for the determination of digestibility coefficients of fish

meal analogue (FMA), fish meal (FM), and soybean meal (SBM). ................................... 156 Table 4 Proximate composition1, phosphorus content1, and amino acid profile1 of the ingredients

used in the growth and digestibility trials. .......................................................................... 157 Table 5 Proximate composition1 (% as is), mineral composition (% as is: phosphorus, sulfur,

potassium, magnesium, calcium, sodium; mg kg-1 as is: iron, manganese, copper, zinc)2 and amino acid profile1 (% as is) of the test diets used in the growth trial 1 and 2. .................. 158

Table 6 Proximate composition1 (% as is), mineral composition (% as is: phosphorus, sulfur,

potassium, magnesium, calcium, sodium; mg kg-1 as is: iron, manganese, copper, zinc)2 and amino acid profile1 (% as is) of the test diets used in the growth trial 3. ........................... 160

Table 7 Performance of juvenile Pacific white shrimp L. vannamei (Initial weight: 0.47, 0.40,

0.25 g in trial 1, 2, and 3, respectively) offered diets with different fish meal analogue (FMA) levels (0, 4.85, 9.70, 14.55, and 19.44%) replacing different levels of fish meal for six weeks. ............................................................................................................................ 162

Table 8 Pearson correlation coefficients of final biomass, final mean weight, weight gain (WG),

feed conversion ratio (FCR), and survival in trial 1 and trial 2 with phosphorus levels of the diets. The first line of each cell is the value of correlation coefficient and the second line of each cell is P-value. ............................................................................................................ 164

Table 9 Whole body proximate composition (% dry weight basis), phosphorus content (P, % dry

weight basis), and protein and phosphorus retention (%) when shrimp were offered diets supplemented with different fish meal analogue levels replacing fish meal without balanced for phosphorus for six weeks in trial 2. ............................................................................... 165

Table 10 Whole body proximate1 composition, mineral composition1, and protein and

phosphorus retention when shrimp were offered diets supplemented with different fish meal

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analogue levels replacing different levels of fish meal balance for phosphorus for six weeks in trial 3. .............................................................................................................................. 166

Table 11 Apparent dry matter (ADM), apparent energy (AED) and apparent protein (APD)

digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. ..................................................................... 167

Table 12 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal

(FM), poultry meal (PM), and fish meal analogue (FMA) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei. .................................................... 168

ChapterVII Table 1 Proximate composition1, phosphorus content1, and amino acid profile1 of the primary

protein sources used in diet formulations. .......................................................................... 204 Table 2 Mineral composition1 of the three batches Ulva meal (UM1, UM2, and UM3) utilized in

feed formulations. ............................................................................................................... 205 Table 3 Proximate1 and mineral1 composition of Ulva meal 2 (UM2) utilized in trial 2 and 3

collected from seven different dates. .................................................................................. 206 Table 4a Formulation of test diets designed to evaluate Ulva meal 1 (UM1) as a replacement for

fish meal on an iso-nitrogenous basis (Trial 1). .................................................................. 207 Table 4b Proximate composition1 and amino acid profile1 of the test diets used in the trial 1. . 208 Table 4c Protein retention efficiency and growth performance of juvenile Pacific white shrimp

(0.26±0.02g) offered diets with different Ulva meal 1 levels (0, 6.35, 12.70, 19.05, and 25.40%) as a fish meal replacement over a six-week growth trial (Trial 1). ...................... 209

Table 4d Proximate3 analysis of whole shrimp body offered varying Ulva meal 1 levels (0, 6.35,

12.70, 19.05, and 25.40%) as a replacement of fish meal over a six-week growth trial (Trial 1). ........................................................................................................................................ 210

Table 5a Formulation of test diets designed to evaluate three batches of Ulva meal (UM1, UM2,

and UM3) as a replacement for soybean meal on an iso-nitrogenous basis (Trial 2). ........ 211 Table 5b Proximate composition1, phosphorus content1, and amino acid profile1 of the test diets

used in the trial 2. ................................................................................................................ 213 Table 5c Mineral profile1 of the test diets used in the trial 2. ..................................................... 214 Table 5d Growth performance of juvenile Pacific white shrimp (0.24±0.01g) offered diets with

different levels of three batches Ulva meal (UM1, UM2, and UM3) for five weeks (Trial 2).............................................................................................................................................. 215

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Table 5e Proximate composition2 and amino acid profile2 of whole shrimp body offered diets

contain different levels of three batches Ulva meal (UM1, UM2, and UM3) levels in trial 2.............................................................................................................................................. 216

Table 5f Protein2 and amino acids3 retention efficiencies of Pacific white shrimp offered varying

Ulva meal 2 levels diets in trial 2 for five weeks. ............................................................... 218 Table 6a Formulation of test diets designed to evaluate Ulva meal 1, 2, and 3 as a replacement

for soybean meal on a digestible protein basis (Trial 3). .................................................... 220 Table 6b Proximate composition1, mineral composition2, and amino acid profile1 of the test diets

used in trial 3. ...................................................................................................................... 222 Table 6c Growth performance of juvenile Pacific white shrimp L. vannamei (Initial weight

0.98g) offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal over six weeks (Trial 3). .......................... 224

Table 6d Proximate composition2 and amino acids profile2 of shrimp at the conclusion of a 6-

week growth trial in which shrimp were offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal (Trial 3). ........................................................................................................................................ 225

Table 6e Protein2 and amino acid3 retention efficiency of Pacific white shrimp at the conclusion

of a 6-week growth trial in which shrimp were offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal (Trial 3). ........................................................................................................................................ 226

Table 7a Formulation of test diets designed to evaluate Ulva meal 4 as a protein source in trial 4

............................................................................................................................................. 227 Table 7b Proximate composition1, mineral composition2 and amino acid profile1 of the test diets

used in trial 4. ...................................................................................................................... 228 Table 7c Performance of juvenile Pacific white shrimp L. vannamei (Initial weight 0.15g)

offered diets formulated to evaluate Ulva meal 4 as a replacement for soybean meal and fish meal on an iso-nitrogen basis in juvenile shrimp over six weeks (Trial 4). ....................... 230

Table 7d Proximate2 composition of shrimp at the conclusion of a 6-week growth trial in which

shrimp were offered diets formulated to evaluate Ulva meal 4 as a replacement for soybean meal and fish meal on an iso-nitrogen basis in juvenile shrimp (Trial 3). .......................... 231

Table 8a Composition of reference diet for the determination of digestibility coefficients of Ulva

meal 1 and 2. ....................................................................................................................... 232

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Table 8b Apparent dry matter (ADM), apparent energy (AED) and apparent protein (APD) digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered to Pacific white shrimp (L. vannamei) ................................................................... 233

Table 8c Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal

(FM), Ulva meal 1 (UM1) and Ulva meal 2 (UM2) using 70:30 replacement technique offered to Pacific white shrimp (L. vannamei) ................................................................... 233

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List of Figures

Figure 1 (a) In trial 1, relationship between weight gain (y) of shrimp and incorporation levels of Ulva meal 1 levels (x) in the diets. The regression line is described by y = 0.1686x3 - 6.2114x2 + 34.409x + 1797.8 (R2 = 0.4922, P < 0.0001). (b) In trial 1, relationship between FCR (y) and supplemental Ulva meal levels (x) in the diets. The regression line is described by y = 0.0276x + 1.7708 (R2 = 0.3582, P = 0.0001). (c) In trial 1, relationship between lipid content (y) of shrimp body and supplemental Ulva meal levels (x) in the diets. The regression line is described by y = -0.0962x + 7.4 (R2 = 0.3503, P = 0.0002)……………………………………………………….241 Figure 2 (a) In trial 2, relationship between weight gain (y) and Ulva meal 2 levels (x) in the diets. The regression line is described by y = 1.1925x2 -62.699x + 1713.1 (R2 = 0.6815, P < 0.0001). (b) In trial 2, relationship between FCR (y) and supplemental Ulva meal 2 levels (x) in the diets. The regression line is described by y = -0.0703x + 1.5451 (R2 = 0.4766, P < 0.0001). (c) In trial 2, relationship between survival (y) and supplemental Ulva meal 2 levels (x) in the diets. The regression line is described by y = -1.1607x + 100.27 (R2 = 0.6113, P < 0.0001). (d) In trial 2, relationship between lipid content (y) of shrimp body and supplemental Ulva meal 2 levels (x) in the diets. The regression line is described by y = 0.0078x2 - 0.4095x + 7.8033 (R2 = 0.9051, P < 0.0001)………………………………………………………………………………………..242 Figure 3 (a) In trial 4, relationship between weight gain (y) of shrimp and incorporation levels of Ulva meal 4 levels (x) in the diets. The regression line is described by y = 2.6848x2 - 119.17x + 3049.3 (R2 = 0.6934, P < 0.0001). (b) In trial 4, relationship between FCR (y) and supplemental Ulva meal 4 levels (x) in the diets. The regression line is described by y = 0.06x + 1.8532 (R2 = 0.721, P < 0.0001). (c) In trial 4, relationship between lipid content (y) of shrimp body and supplemental Ulva meal 4 levels (x) in the diets. The regression line is described by y = -0.1903x + 7.9555 (R2 = 0.7224, P < 0.0001)…………………………………………………………….243

1

CHAPTER I

Introduction

Pacific white shrimp, Litopenaeus vannamei is native to the Eastern Pacific coast from

Gulf of California, Mexico to Tumbes, North of Peru (PeÂrez Farfante and Kensley, 1997). It is

well known for its ability to grow and survive well under a wide range of environmental and

artificial culture conditions, Adaptability to commercial culture has made it the primary cultured

shrimp species as well as atop aquaculture commodity (Roy et al., 2009). The production of

Pacific white shrimp increased from 0.15 million metric tons in 2000 to 4.5 million metric tons

in 2016 (Anderson, 2016), and was estimated to increase to 9.2 million tonnes in 2020 (Tacon

and Metian, 2008). The USA was the top global import market for imported shrimp in 2015,

with 0.59 million tonnes imported, and the import value reached 5.46 billion dollars in 2015

(FAO, 2015).

Feed represents one of the largest variable costs associated with fed culture systems;

reducing feed costs can produce considerable savings (Davis and Sookying, 2009).

Manufacturing of marine shrimp feed uses 24% to 27% of the world’s fish meal (Tacon and

Metian, 2008) making them one of the prime consumers. Commercial shrimp feed formulations

historically contain from 25% to 50% fish meal, which represents the primary and most

expensive protein ingredient (Dersjant-Li, 2002; Tacon and Metian, 2008). Fish meal (FM) is

preferred among other protein sources because it is an excellent source of essential nutrients such

as protein and indispensable amino acids, essential fatty acids, cholesterol, vitamins, minerals

attractants and unidentified growth factors (Samocha et al., 2004; Swick et al., 1995). The cost of

FM has increased over time because of increased demand, limitations of availability, and

growing social and environmental concerns regarding wild fish extraction practices (Tacon and

2

Metian, 2008). Due to limited supply and increasing prices, we must shift our emphasis and use

this ingredient only when nutrient requirements of the animal demand its use (Davis and

Sookying, 2009).

To develop sustainable and environmentally friendly aquaculture, various terrestrial

plant-based ingredients that contain high protein content are potential alternative sources for fish

meal (NRC, 2011). Terrestrial plant-based protein sources such as soybean meal, canola meal,

corn gluten meal are available worldwide and have a relatively low cost compared to fish meal.

Among plant protein sources, soybean meal (SBM) is the most abundant and has received

considerable attention as a replacement for fish meal in aquatic animal feeds because of its

availability, balanced amino acid profile and consistent composition (Akiyama, 1989; Akiyama

et al., 1991; Amaya et al., 2007; Hardy, 1999; Samocha et al., 2004; Swick et al., 1995; Tacon,

2000). However, the presence of anti-nutritional factors (ANF) such as lectins, oligosaccharides,

saponins, and trypsin inhibitors, can limit the inclusion levels of SBM in some aquaculture feeds.

Moreover, as the popularity of SBM as a feed ingredient increased, its price continued to

increase from 185.02 dollars per metric ton in 2001 to 443.41 dollars per metric ton in 2016. In

addition, application of terrestrial plant protein such as SBM in aquaculture feeds may affect the

food costs for human communities in developing countries (Delgado et al., 2002; 2003).

As shrimp culture has become an expanded and intensified economic activity, the

demand for more cost-effective and sustainable protein sources continues to increase. In this

instance, incorporation of marine plant protein (i.e. macro-algae) in aquaculture feeds would be

an alternative strategy to reduce the reliance on FM and terrestrial plant protein sources such as

SBM. The chemical composition of macro-algae can be influenced by both physical and

chemical factors such as temperature, salinity, light (Lobban and Harrison, 1994) and nutrient

3

concentrations (Björnsäter and Wheeler, 1990; Floreto et al., 1996; García‐Ferris et al., 1996)

during cultivation. Therefore, the product quality including protein content, lipid content, and

tissue pigmentation of macro-algae can be somewhat manipulated by controlling the main

parameters of cultivation (i.e. nutrient loading, stocking density, mixing regime, etc.) (Guerin

and Bird, 1987; Neori, 1996; Shpigel et al., 1999). Nitrogen-enriched conditions like the

effluents of fish or shrimp farms, where seaweeds are used as bio-filters, can enhance their

protein contents (Lahaye et al., 1995; Pinchetti et al., 1998). Lipid content can be raised by

manipulations of nutrient supply and other growth parameters. Macro-algae are also rich sources

of minerals (7% to 38% dry weight basis) with a broad mineral composition including Bo, Ca,

Cl, Co, Cu, F, Fe, I, K, Mg, Mn, Mo, Na, Ni, P, S, Se, Zn.

Macro-algae can benefit from recycling waste carbon dioxide (CO2) from combustive

energy production (that otherwise pollutes the atmosphere) and waste nutrients produced by

intensive aquaculture operations, agriculture, intensive animal operations, municipal waste

treatment, and the like (Kaushik and Troell, 2010; Sargent and Tacon, 1999). In an integrated

cultivation system, the macro-algae use the metabolic residues of animals as nutrients, absorb

CO2 and produce O2 for the environment (Marinho-Soriano et al., 2008). The interaction allows

the excretion of an organism to serve as food for another (Qian et al., 1996). Presently,

significant improvements in growth and survival rate have been observed when Pacific white

shrimp, Litopenaeus vannamei (Cruz-Suárez et al., 2010), tiger shrimp, Penaeous monodon Fabr

(Izzati, 2012; Tsutsui et al., 2010), and yellowtail shrimp, Farfantepenaeus californiensis

(Portillo‐Clark et al., 2012) are co-cultured with macro-algae.

Macro-algae have been demonstrated to replace small fractions (1 to 4%) of FM content

in diets of Sea bass, Dicentrarchus labrax (Valente et al., 2006), Rainbow trout, Oncorhynchus

4

mykiss (Soler-Vila et al., 2009; Yıldırım et al., 2009) and Pacific white shrimp (Rodríguez-

González et al., 2014). Growth responses when high levels of algae are used to replace FM

content in diets of aquatic animals vary. Xu et al. (2011) reported that weight gain of teleost fish,

Siganus canaliculatus was significantly decreased when using seaweed Gracilaria

lemaneiformis to replace 10% FM. However, Stadtlander et al. (2013) indicated that 7.5 and 15%

FM replacement by red alga Nori Porphyra yezoensis Ueda did not significantly affect the

growth performance of Nile tilapia, Oreochromis niloticus.

Besides macro-algae, bacteria biomass is another potential feed ingredient as a

replacement for FM and SBM in aquaculture feeds. Rapid growth and high protein content are

well known properties of bacteria in protein production (Anupama and Ravindra, 2000; Kuhad et

al., 1997; Stringer, 1982). Methane, the main component of natural gas, which is found widely in

nature (Hanson and Hanson, 1996; Dalton, 2005) is an attractive substrate for bacterial protein

production. The abundant supply, cheap transportation, and reasonable cost of natural gas,

indicate that protein production from natural gas could be realistic on a large scale (Overland et

al., 2010).

The naturally occurring methanotroph Methylococcus capsulatus (Bath) has shown high

efficiency in production of bacterial protein from methane (Bothe et al., 2002). Considerable

researches have been carried out on the bacterial meal, produced from mainly methane by natural

gas fermentation, as a protein source for a number of fish species, including Atlantic salmon,

Salmo salar (Aas et al., 2006a; Berge et al., 2005), rainbow trout, Oncorhynchus mykiss (Aas et

al., 2006b; Kiessling and Askbrandt, 1993; Storebakken et al., 2004), and Atlantic halibut,

Hippoglossus hippoglossus (Aas et al., 2007), and Florida pompano, Trachinotus carolinus

(Melanie et al., 2015). However, the growth responses of the fish in these studies to the bacterial

5

meal are not consistent, which may due to the different fish species and various bacterial strains.

Information about the nutrient digestibility values of bacterial meal is still limited. However,

information about protein digestibility of the diets contained bacterial meal is available.

Storebakken et al., 1998 reported that the apparent protein, fat and energy digestibility of the diet

that contained 20% bacterial meal replacing 20% FM were 89.7%, 88.3%, and 82.1%,

respectively, which were close to those of FM-based diet (91.5%, 90%, and 86%, respectively).

Given the great potential of the alternative ingredients provided above, it is important for

us to explore the nutritional value of these products in order to ensure sustainability and options

for feed formulator. Therefore the purpose of this research is to evaluate the utilization of

potential alternative ingredient in practical diets for Pacific white shrimp, L. vannamei.

6

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Oil Chemists Society, Champaign, Illinois, USA, pp. 252-265.

Akiyama, D.M., Dominy, W., Lawrence, A.L., 1991. Penaeid shrimp nutrition for the

commercial feed industry: Revised, Proceedings of the Aquaculture Feed Processing and

Nutrition Workshop, Thailand and Indonesia, September, pp. 19-25.

Amaya, E.A., Davis, D.A., Rouse, D.B., 2007. Replacement of fish meal in practical diets for the

Pacific white shrimp (Litopenaeus vannamei) reared under pond conditions. Aquaculture

262, 393-401.

Björnsäter, B.R., Wheeler, P.A., 1990. Effect of nitrogen and phosphorus supply on growth and

tissue composition of Ulva fenestrata and Enteromorpha intestinalis (ulvales,

chlorophyta) 1. Journal of Phycology. 26, 603-611.

Cruz-Suárez, L.E., León, A., Peña-Rodríguez, A., Rodríguez-Peña, G., Moll, B., Ricque-Marie,

D., 2010. Shrimp/Ulva co-culture: a sustainable alternative to diminish the need for

artificial feed and improve shrimp quality. Aquaculture. 301, 64-68.

Davis, D.A., Sookying, D., 2009. Strategies for reducing and/or replacing fishmeal in production

diets for the Pacific white shrimp, Litopenaeus vannamei. The rising tide 108-114.

Delgado, C.L., Rosegrant, M.W., Meijer, S., Wada, N., and Ahmed, M., 2002. Fish as Food:

Projections to 2020.

Delgado, C.L., Wada, N., Rosegrant, M.W, Siet Meijer, S., and Ahmed, M., 2003. Outlook for

Fish to 2020, Meeting Global Demand. A 2020 Vision for Food, Agriculture, and the

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Environment Initiative, International Food Policy Research Institute, Washington,D.C.,

U.S.A. World Fish Center, Penang, Malaysia, 33 pages.

Dersjant-Li, Y., 2002. The use of soy protein in aquafeeds. Avances en Nutricion Acuicola VI.

Memorias del VI Simposium Internacional de Nutricion Acuicola. 3, 541-558.

Floreto, E.A.T., Teshima, S., Ishikawa, M., 1996. Effects of nitrogen and phosphorus on the

growth and fatty acid composition of Ulva pertusa Kjellman (Chlorophyta). Botanica

marina. 39, 69-74.

García‐Ferris, C., Ríos, A., Ascaso, C., Moreno, J., 1996. Correlated biochemical and

ultrastructural changes in nitrogen‐starved euglena gracilis1. Journal of phycology. 32,

953-963.

Guerin, J.M., and Bird, K.T., 1987. Effects of aeration period on the productivity and agar

quality of Gracilaria sp. Aquaculture, 64(2):105-110.

Hardy, R.W., 1999. Aquaculture’s rapid growth requirements for alternate protein sources. Feed

Management 50, 25-28.

Izzati, M., 2012. The role of seaweeds Sargassum polycistum and Gracilaria verrucosa on

growth performance and biomass production of tiger shrimp (Penaeous Monodon Fabr).

Journal of Coastal Development. 14, 235-241.

Kaushik, S., Troell, M., 2010. Taking the fish-in fish-out ratio a step further. Aquac. Eur.

35(1):15-17.

Lahaye, M., Gomez‐Pinchetti, J.L., del Rio, M.J., Garcia‐Reina, G., 1995. Natural decoloration

composition and increase in dietary fibre content of an edible marine algae, Ulva rigida

(Chlorophyta), grown under different nitrogen conditions. Journal of the Science of Food

and Agriculture. 68, 99-104.

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Lobban, C.S., Harrison, P.J., 1994. Seaweed ecology and physiology. Cambridge University

Press.

Neori, A., 1996. The form of N-supply (ammonia or nitrate) determines the performance of

seaweed biofilters integrated with intensive fish culture. Israeli J. Aquacult. Bamidgeh

48:19-27.

NRC, 2011. Nutrient requirements of fish and shrimp. National academies press.

PeÂrez Farfante, I., Kensley, B., 1997. Penaeoid and sergestoid shrimps and prawns of the

world. Keys and diagnoses for the families and genera. MeÂm Mus natn Hist Nat, Paris

175, 1-79.

Pinchetti, J.L.G., del Campo Fernández, E., Díez, P.M., Reina, G.G., 1998. Nitrogen availability

influences the biochemical composition and photosynthesis of tank-cultivated Ulva rigida

(Chlorophyta). Journal of Applied Phycology. 10, 383-389.

Portillo‐Clark, G., Casillas‐Hernández, R., Servín‐Villegas, R., Magallón‐Barajas, F.J., 2012.

Growth and survival of the juvenile yellowleg shrimp Farfantepenaeus californiensis

cohabiting with the green feather alga Caulerpa sertularioides at different temperatures.

Aquaculture Research. 44, 22-30.

Samocha, T.M., Davis, D.A., Saoud, I.P., DeBault, K., 2004. Substitution of fish meal by co-

extruded soybean poultry by-product meal in practical diets for the Pacific white shrimp,

Litopenaeus vannamei. Aquaculture 231, 197-203.

Sargent J.R. and Tacon A.G., 1999. Development of farmed fish: a nutritionally necessary

alternative to meat. Proc. Nutr. Soc. 58:377-83.

9

Shpigel, M., Ragg, N.L., Lupatsch, I., Neori, A., 1999. Protein content determines the nutritional

value of the seaweed Ulva lactuca L for the abalone Haliotis tuberculata L. and H. discus

hannai INO. J. Shellfish Res. 18:227-233.

Soler-Vila, A., Coughlan, S., Guiry, M.D., Kraan, S., 2009. The red alga Porphyra dioica as a

fish-feed ingredient for rainbow trout (Oncorhynchus mykiss): effects on growth, feed

efficiency, and carcass composition. Journal of Applied Phycology. 21, 617-624.

Sororzano, L., 1969. Determination of ammonia in natural waters by the phenolhypochlorite

method. Limnol Oceanogr. 14, 799-801.

Spotte, S., 1979. Fish and invertebrate culture: water management in closed systems.

Stadtlander, T., Khalil, W.K.B., Focken, U., Becker, K., 2013. Effects of low and medium levels

of red alga Nori (Porphyra yezoensis Ueda) in the diets on growth, feed utilization and

metabolism in intensively fed Nile tilapia, Oreochromis niloticus (L.). Aquaculture

Nutrition. 19, 64-73.

Swick, R.A., Akiyama, D.M., Boonyaratpalin, M., Creswell, D.C., 1995. Use of soybean meal

and synthetic methionine in shrimp feed. American Soybean Association. Technical

Bulletin.

Tacon, A.G.J., 2000. Rendered animal by-products: a necessity in aquafeeds for the new

millennium. Global Aquaculture Advocate 3, 18-19.

Tacon, A.G.J., Metian, M., 2008. Global overview on the use of fish meal and fish oil in

industrially compounded aquafeeds: trends and future prospects. Aquaculture 285, 146-

158.

Tsutsui, I., Kanjanaworakul, P., Srisapoome, P., Aue-umneoy, D., Hamano, K., 2010. Growth of

giant tiger prawn, Penaeus monodon Fabricius, under co-culture with a discarded

10

filamentous seaweed, Chaetomorpha ligustica (Kützing) Kützing, at an aquarium-scale.

Aquaculture international. 18, 545-553.

Valente, L.M.P., Gouveia, A., Rema, P., Matos, J., Gomes, E.F., Pinto, I.S., 2006. Evaluation of

three seaweeds Gracilaria bursa-pastoris, Ulva rigida and Gracilaria cornea as dietary

ingredients in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture. 252, 85-

91.

Xu, S., Zhang, L., Wu, Q., Liu, X., Wang, S., You, C., Li, Y., 2011. Evaluation of dried seaweed

Gracilaria lemaneiformis as an ingredient in diets for teleost fish Siganus canaliculatus.

Aquaculture International. 19, 1007-1018.

Yıldırım, Ö., Ergün, S., Dernekbaşı, S.Y., Türker, A., 2009. Effects of two seaweeds (Ulva

lactuca and Enteremorpha linza) as a feed additive in diets on growth peformance, feed

utilization and body composition of rainbow trout (Oncorhynchus mykiss).

11

CHAPTER II

EVALUATION OF FLASH DRIED YEAST AS A NUTRITIONAL SUPPLEMENT IN

PLANT BASED PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus

vannamei

1. Introduction

As shrimp culture has become an expanded and intensified economic activity, bacterial

and viral diseases are considered to threaten the further progress of semi-intensive and intensive

shrimp culture (Pérez-Sánchez et al. 2014). Antibiotics have been utilized to supplement in the

shrimp feeds for prevention and treatment of diseases (Cabello 2006; Taylor et al. 2011).

However, the use of antibiotics may develop bacterial strains that are more resistant to antibiotic

treatment, meanwhile the antibiotic residues in cultured animals may pose negative impacts on

human health (Sharifuzzaman & Austin 2009). To tackle disease problems and avoid potential

disadvantages of antibiotics, a number of alternative strategies such as the use of vaccine,

immunostimulants, probiotics, and prebiotics have received considerable attention (Li & Gatlin

2005).

The probiotics are usually members of the healthy microbiota associated with the host

(Pérez‐Sánchez et al. 2014). They can prevent bacterial diseases by producing inhibitory

compounds to create a hostile environment for pathogens, competing for essential nutrients and

adhesion sites or modulating the immune response (Merrifield et al. 2010). Probiotic effects in

aquaculture are not only limited to intestinal tract of aquatic animals, but also can improve the

health of the host by controlling pathogens and improving water quality (Verschuere et al. 2000;

Zheng et al. 2012).

12

Yeast is one of the probiotics, which is commonly used in aquaculture either alive to feed

live food organisms, or after processing, as a feed ingredient (Stones & Mills 2004). Yeast cells

contain β-glucans, nucleic acid, oligosaccharides, as well as polyamines, which may help to

improve the immune response and growth performance as well as metabolism in fish and shrimp

(Gatesoupe 2007). Yeasts are frequently used as a dietary supplement in fish and shrimp feeds to

increase growth performance, feed intake, survival and disease resistance (Deng et al. 2013; Essa

et al. 2011; Hoseinifar et al. 2011; Li & Gatlin 2003; 2004; 2005; Sheikhzadeh et al. 2012; Shen

et al. 2010; Vechklang et al. 2012; Yang et al. 2010) as well as an alternative protein sources for

fishmeal (Gause & Trushenski 2011a; b; Hauptman et al. 2014; Lunger et al. 2006; Oliva-Teles

& Gonçalves 2001; Peterson et al. 2012).

The two common yeasts that used in aquaculture feeds are brewers yeast Saccharomyces

cerevisiae (BY), which is a natural product of the brewing industry and grain distillers dried

yeast (GDDY), which is a co-product obtained from the bioethanol industry. The yeast product

utilized in the present study is flash dried yeast (FDY) that is a novel product produced by low

pH fermentation of Saccharomyces Pombe.

Albeit there are some studies demonstrating positive effects of various yeast supplements

in Pacific white shrimp diets on the disease tolerance and growth performance, there is limited

data in apparent digestibility coefficients of yeast products. Hence, The objectives of this project

was to determine the growth response of Pacific white shrimp juveniles to increasing FDY levels

in a soybean meal based feed formulation and determined apparent digestibility values for yeast

as compared to other protein sources.

13

2. Materials and Methods

2.1 Experimental diets

All test diets were formulated to be isonitrogenous and isolipidic (350 g kg-1 protein and

80 g kg-1 lipid). For the growth trial, five experimental diets were formulated. The first diet or

basal diet did not contain FDY. Whereas the next four diets contained FDY at increasing levels

(10, 20, 40, and 60 g kg-1) (Table 1). The reference diet (Table 4) for ingredient digestibility trial

was formulated to include 10 g kg-1 chromic oxide as inert marker. Test diets were made using a

70:30 mixture of the reference diet and test ingredients.

Primary ingredients (Table 2) were analyzed for proximate composition in the diets

formulated. Pre-ground dry ingredient and oil were mixed in a food mixer (Hobart Corporation,

Troy, OH, USA) for 15 min. Hot water was then blended into the mixture to obtain a consistency

appropriate for pelleting. Diets were pressure-pelleted using a meat grinder with a 3-mm die, air-

dried (< 50 °C) to a moisture content of 80-100 g kg-1. Pellets were crumbled, packed in sealed

plastic bags and stored in a freezer until needed. The diets were analyzed at University of

Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA) for

proximate composition (g kg-1 as is) and amino acid profile (% as is) (Table 3).

2.2 Growth trial

The growth trial utilized 5 treatments with 5 replicates in each treatment. It was

conducted in a semi-closed recirculation system. Juvenile shrimp were obtained from the nursery

system and selected by hand-sorting to a uniform size. Juvenile shrimp (initial weight 1.78±0.03

g) were stocked into 25 tanks with 10 shrimps in each aquarium (80 L). A sub-sample of shrimp

from the initial stocking was retained for whole body samples to be utilized for later protein

14

retention analysis. As shrimp are difficult to handle, intermittent weights were not taken.

However, shrimp were counted to readjust daily feed input on a weekly basis. Based on

historical results, a fixed ration was calculated assuming a 1.8 feed conversion ratio and 0.8 to

1.4 g week-1. Consequently, for each tank a fixed ration of 2.31 g day-1 for the first week, 2.83 g

day-1 for the second week, 2.90 g day-1 for the third week, and 3.09 g day-1 for the forth week,

3.34 g day-1 for the fifth week, and 3.6 g day-1 for the six week was offered over 4 feedings.

Dissolved oxygen (DO), temperature, and salinity were measured twice daily by using a

YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA). pH was measured twice

weekly by using a waterproof pHTestr30 (Oakton instrument, Vernon Hills, IL, USA). Water

samples were taken to measure total ammonia-nitrogen (TAN) and nitrite every week. TAN and

nitrite were determined by using the methods described by Solorzano (1969) and Spotte (1979),

respectively. During the experiment period DO, temperature, salinity, pH, TAN, and nitrite were

maintained within acceptable ranges for L. vannamei at 5.36±0.26 mg L-1, 29.0±1.2 °C, 13.1±0.3

g L-1, 7.45±0.33, 0.066±0.0048 mg L-1, and 0.040±0.037 mg L-1, respectively.

Shrimps were counted to readjust daily feed input on a weekly basis. At the conclusion of

6-week growth trial, shrimps were counted and group weighted. Mean final weight, feed

conversion ratio (FCR), weight gain (WG), biomass, and survival were determined. After

obtaining the final total weight of shrimps in each aquarium, 4 shrimps were randomly selected

and frozen at -20 °C for subsequent determination of whole body composition. Proximate

composition of whole shrimp was analyzed by University of Missouri-Columbia, Agriculture

Experiment Station Chemical Laboratory (Columbia, MO, USA). Protein retention was

calculated as follows:

15

Protein retention (%) = (final weight × final protein content) - (initial weight × initial protein

content) × 100 / protein offered.

2.3 Digestibility trial

The digestibility trial was conducted in the mentioned recirculation system and utilized

six shrimp per aquaria with six aquaria per dietary treatment. Once acclimated for three days to

the test diets, feces from two aquaria were pooled (n=3) and collected over a five-day period or

until adequate samples were obtained. To obtain fecal samples, the aquaria were cleaned by

siphoning before each feeding with the first collection of the day discarded. After cleaning, the

shrimp were offered an excess of feed and then about 1 hour later feed was removed and feces

were collected by siphoning onto a 500 µm mesh screen. Collected feces were rinsed with

distilled water, dried at 105 °C until a constant weight was obtained, and then stored in freezer

(−20 °C) until analyzed. Apparent digestibility coefficients for dry matter, protein, energy, and

amino acids were determined by using chromic oxide (Cr2O3, 10 g kg -1) as an inert marker.

Chromium concentrations were determined by the method of McGinnis and Kasting (1964) in

which, after a colorimetric reaction, absorbance is read on a spectrophotometer (Spectronic

genesis 5, Milton Roy Co., Rochester, NY, USA) at 540 nm. Gross energy of diets and fecal

samples were analyzed with a Semi micro-bomb calorimeter (Model 1425, Parr Instrument Co.,

Moline, IL, USA). Protein of diets and fecal samples were determined by micro-Kjeldahl

analysis (Ma and Zuazaga, 1942). Amino acids were analyzed by University of Missouri-

Columbia, Agriculture Experiment Station Chemical Laboratory. The apparent digestibility

coefficient of dry matter (ADMD), protein (ADP), energy (ADE), and amino acids (ADAA)

were calculated according to Cho et al. (1982) as follows:

16

ADMD (%)= 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces)]

ADP, ADE, and ADAA (%)=100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces) × (% nutrient in

feces / % nutrient in feed]

The apparent digestibility coefficients of the test ingredients for dry matter, energy,

protein and amino acids were calculated according to Bureau & Hua (2006) as follows:

ADCtest ingredient = ADCtest diet + [(ADCtest diet – ADCref. diet) × (0.7 × Dref / 0.3 × Dingr)]

where Dref = % nutrient (or KJ/g gross energy) of reference diet mash (as is); Dingr = % nutrient

(or KJ/g gross energy) of test ingredient (as is).

2.4 Statistical analysis

All the data were analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). Data from

the growth and digestibility trial were analyzed using one-way ANOVA to determine significant

differences (P<0.05) among treatments followed by the Tukey’s multiple comparison test to

determine difference between treatments. Arcsine square root transformation was used prior to

analysis for the proportion data in growth and digestibility trial. False discover rate (FDR)

controlling procedures were used to adjust the P-value in order to control the FDR for the amino

acids digestibility data.

3. Results

Performances and protein retention efficiency of Pacific white shrimp, L. vannamei

offered diets contained different FDY levels are presented in Table 5. Final biomass, final mean

weight, WG, FCR, and PRE of Pacific white shrimp were not significantly influenced when

17

FDY was utilized up to 40 g kg-1 of the diet. However, significantly reduced growth, feed

utilization, and PRE were observed when FDY was supplemented at 60 g kg-1 feed.

Proximate analysis of whole shrimp body offered diets with FDY levels are presented in

Table 6. Supplementation of FDY in the practical diets of Pacific white shrimp did not affect

protein (791.9 to 810.9 g kg-1), moisture (767.5 to 776.4 g kg-1), lipid (50.0 to 65.3 g kg-1), crude

fiber (52.8 to 56.9 g kg-1), and ash (119.9 to 124.7 g kg-1) content of whole shrimp body.

ADMD, ADE, and ADP for the diet (D) and ingredient (I) using 70:30 replacement

technique offered to Pacific white shrimp are presented in Table 7. The digestibility trial

contained a range of ingredients; hence, we have provided a few other ingredients as a reference.

The analyzed proximate composition of FDY as compares to fishmeal was lower in total protein

and lipid content (Table 2). In order to confirm the results, fecal samples for basal diet and FM

diet were recollected. FM1 and FM2 represent the first collection and second collection,

respectively. Basal diet 1 and Basal diet 2 represent first collection and second collection basal

diet, respectively. The results turned out to be quite similar, which indicated that the feces

collection and samples analysis methods we utilized in the digestibility study are consistent. The

energy and protein digestibility of FDY were 38.20% and 53.47%, respectively, which are

significantly lower than fishmeal (FM) and soybean meal (SBM).

Apparent amino acids (AA) digestibility values for the SBM, FM, and FDY using 70:30

replacement technique offered to Pacific white shrimp are presented in Table 8. The analyzed

amino acids composition of FDY as compared to FM was lower in concentrations of individual

amino acids especially the two common limiting amino acids (methionine and lysine) for Pacific

white shrimp (Table 2). Apparent digestibility coefficients of alanine, arginine, aspartic acid,

cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,

18

serine, threonine, tryptophan, tyrosine, valine and total amino acids of FDY were similar to those

of FM but significantly lower than those of SBM. Glycine and hydroxylysine digestibility of

FDY were significantly lower than both those of FM and SBM.

4. Discussion

Yeast is a feed ingredient that can originate from a wide range of sources and has shown

its potential to be used as nutritional supplement and protein source in production diets

(Achupallas et al. 2015; Gause & Trushenski 2011a). The digestibility of a feed ingredient can

provide estimates of nutrient availability in the ingredient, which helps to select ingredients that

optimize the nutritional value and cost of the formulated feed (Brunson et al. 1997).

Unfortunately, nutrient digestibility may show a high inconsistency due to the feeding practices,

environmental conditions, feed processes as well as diet digestibility approaches (Brunson et al.

1997). Protein digestibility of FM in the current study for the two collected samples (FM1 and

FM2) are 67.07% and 71.30%, respectively. Terrazas-Fierro et al. (2010) reported that protein

digestibility of various FMs for Pacific white shrimp ranged from 62.7% to 84.9%. Brunson et

al. (1997) indicated that protein digestibility of FM for white shrimp Penaeus setiferus is

75.85%. The protein availability of FM in the current study falls into the range of protein

digestibility of FMs tested by other researchers. The similar results of basal diet and FM diet

from the two fecal samples collection points to consistency in the feces collection and sample

analysis methods. With regards to the protein digestibility of SBM, it was tested to be 97.03%,

which falls into the ranges of protein digestibility (80.3% to 98.3%) of various SBMs for Pacific

whites shrimp (Cruz-Suarez et al. 2009; Fang et al. 2015; Liu et al. 2012; Yang et al. 2009; Zhou

et al. 2015).

19

In the present study, protein and energy digestibility of FDY were significantly lower

than those of FM and SBM. Amino acids digestibility of FDY was lowest among the three

ingredients tested. Yeast is not typically utilized as protein source in the production diets, hence

only a few studies have evaluated the digestibility values of the yeasts. Hauptman et al. (2014)

reported that protein digestibility (97.6%) of GDDY for rainbow trout Oncorhynchus mykiss was

similar to anchovy fishmeal (97%), soy protein concentrate (99%) and wheat gluten meal

(100%), however, digestibility coefficients for dry matter and energy of GDDY were

significantly lower than those of fishmeal which most likely due to the relatively high nitrogen

free extract content of the ingredient. Amino acids digestibility of the GDDY for rainbow trout O.

mykiss were lower than those of menhaden fishmeal (Hauptman et al. 2014). Rumsey et al.

(1991) indicated that energy and protein digestibility of intact BY and its fractions for rainbow

trout O. mykiss ranged from 62.6 to 77.9% and 63.2 to 84.7%, respectively. Energy and protein

digestibility coefficients of FDY for Pacific white shrimp were lower compared to digestibility

coefficients of GDDY and BY for rainbow trout, which most likely due to the different species

of aquatic animals and various kinds of yeasts utilized in the experiments.

Yeast can serve as a source of dietary nucleotides, which have been shown to stimulate

the immune response, metabolism, and growth in aquatic animals (Gatesoupe 2007). Dietary

yeast supplementation at low levels (no more than 20 g kg-1) as a nutritional supplement in

aquatic animals diets has been demonstrated to improve the growth performance and immune

response in many aquaculture species including African catfish Clarias gariepinus (Essa et al.

2011), hybrid striped bass Morone chrysops × M. saxatilis (Li & Gatlin 2004; 2005), rainbow

trout O. mykiss (Sheikhzadeh et al. 2012), channel catfish Ictalurus punctatus (Li et al. 2011),

20

beluga Huso huso (Hoseinifar et al. 2011), and Pacific white shrimp L. vannamei (Deng et al.

2013; Yang et al. 2010).

With regards to the present growth trial, WG, FCR, and survival of Pacific white shrimp

were not significantly influenced when FDY was utilized up to 40 g kg-1 of the diet. However,

significantly reduced WG and FCR were detected when FDY was incorporated at 60 g kg-1 feed.

The results of present study are in agreement with Li & Gatlin (2003), who reported that WG,

feed efficiency and survival of hybrid stripped bass (Morone chrysops × M. saxatilis) were not

significantly affected when brewer yeast was utilized up to 40 g kg-1 of the diet. Similarly,

Vechklang et al (2012) indicated that WG, feed intake, and survival of Nile tilapia Oreocbromis

niloticus were not significantly influenced when brewer yeast or GroBioti-A were utilized up to

20 g kg-1 of the diet. By contrast, the use of GDDY at the inclusion level up to 300 g kg-1 could

be utilized in diets without causing adverse effects on growth performance of Pacific white

shrimp L. vannamei in a clear water system without natural foods (Achupallas et al. 2016). Also,

Achupallas et al. (2015) demonstrated that in a pond and outdoor green water trial growth

performance of Pacific white shrimp L. vannamei was not significantly affected when GDDY

was used up to 150 g kg-1 feed. GDDY has the potential to be utilized as a protein source and

included at a high level in the diets, which has been confirmed by Gause & Trushenski (2011a;b)

that GDDY at the inclusion level up to 413.3 g kg-1 could be used in diets without causing

negative impacts on the growth performance of sunshine bass and Hauptman et al. (2014) that

WG and FCR of rainbow trout O. mykiss was not affected when GDDY was used up to 112.0 g

kg-1 of the diet.

Variations in the outcome of present study and study conducted by Achupallas et al.

(2016) and Achupallas et al. (2015) may result from different yeasts types. In the present

21

digestibility trial, the protein digestibility of FDY is significantly lower than protein digestibility

of soybean meal. However, the differences of the digestible protein levels between the basal diet

and the diet contains 60 g kg-1 FDY is around 10 g kg-1, which cannot result in the reduced

growth performance of Pacific white shrimp. Also amino acids levels in all the diets (Table 2)

are above the requirements of Pacific white shrimp, thus amino acids deficiency should not be

the factor that caused the negative impact on growth response in the current study. As the

inclusion levels of FDY increased in the diet formulation, we observed the stickiness of the diets

increased during the extrusion, which may increase digesta viscosity and reduce palatability of

the experimental diets. Another possible reason for the reduced growth response may because the

over stimulation of immune response as the inclusion levels of FDY increased.

PRE was not significantly affected when FDY was utilized up to 40 g kg-1 of the diet in

the present study. However, significantly reduced PRE was observed when FDY supplemented

at 60 g kg-1 feed. Hauptman et al. (2014) reported that no significant differences were observed

in PRE when rainbow trout fed with diets contain up to 295.0 g kg-1 GDDY. By contrast, PRE of

sea bass Dicentrarchus labrax was significantly improved when BY was supplemented up to 548

g kg-1 in the diet (Oliva-Teles & Gonçalves 2001). PRE was determined by a number of factors

including dietary protein levels, feed intake, final weight and initial weight of animals as well as

the final and initial protein content of animals (Halver & Hardy 2002). In the current study, no

significant differences were found in dietary protein levels, feed offered to the shrimp, and

protein content of whole shrimp body. The significantly reduced PRE should be caused by the

decreased weight gain of shrimp in the current study.

In the current study, no significant differences were observed in whole body composition

(moisture, protein, lipid, ash, crude fiber) of Pacific white shrimp when fed with diets contain up

22

to 60 g kg-1 FDY. Similarly, Li & Gatlin (2003) reported that whole body composition of

juvenile stripped bass was not affected when brewers yeast was utilized up to 40 g kg-1 of the

diet. Vechklang et al. (2012) indicated that no significant differences were observed in whole

body composition (moisture, protein, lipid and ash) of tilapia fed with diets supplemented with

10 and 20 g kg-1 BY. In addition, many other studies also reported that dietary yeast

supplementation had no significant effects on whole body composition of fish (Essa et al. 2011;

Hauptman et al. 2014; Hoseinifar et al. 2011; Li et al. 2005; Sheikhzadeh et al. 2012). However,

it should be noted that in the present study lipid content in the whole shrimp body decreased

from 65.3 g kg-1 to 50.0 g kg-1 on a dry weight basis (P=0.0794) as the FDY supplementation

levels increased from 0 to 60 g kg-1, which indicates that FDY may have a potential to reduce

lipid content of Pacific white shrimp.

5. Conclusion

The energy and protein digestibility of flash dried yeast are significantly lower than FM

and SBM. Amino acids digestibility of FDY was lowest among the three ingredients tested. With

regards to the growth trial, the use of FDY at 60 g kg-1 caused significant negative impacts on

growth, feed conversion ratio and protein retention. Dietary flash dried yeast supplementation in

the practical diets for Pacific white shrimp had no effects on the proximate composition of the

whole shrimp body. Based on these results, further research regarding the effects of the low

levels (< 40 g kg-1) inclusion of FDY in practical diets on immune responses of Pacific white

shrimp is warranted.

23

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29

Table 1 Formulation and chemical composition of test diets used in the growth trial.

Ingredient (As is basis g kg-1 feed) Diet code

D1 D2 D3 D4 D5

Menhaden fish meal1 60.0 60.0 60.0 60.0 60.0

Soybean meal2 472.0 463.8 455.6 439.5 423.2

Corn protein concentrate3 60.0 60.0 60.0 60.0 60.0

Whole wheat4 280.0 280.0 280.0 280.0 280.0

Flash dried yeast10 0.0 10.0 20.0 40.0 60.0

Menhaden fish oil2 55.7 54.9 54.1 52.6 51.0

Trace mineral premix5 5.0 5.0 5.0 5.0 5.0

Vitamin premix6 18.0 18.0 18.0 18.0 18.0

Choline chloride4 2.0 2.0 2.0 2.0 2.0

Stay C7 1.0 1.0 1.0 1.0 1.0

Mono-dicalcium Phosphate8 18.0 18.0 18.0 18.0 18.0

Lecithin9 10.0 10.0 10.0 10.0 10.0

Cholesterol4 0.5 0.5 0.5 0.5 0.5

Corn starch4 17.8 16.8 15.8 13.4 11.3 1 Omega Protein Inc., Houston, TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g kg-1 premix): Thiamin.HCl, 4.95; Riboflavin, 3.83; Pyridoxine.HCl, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA. 10 Archer Daniels Midland Company, Chicago, IL, USA.

30

Table 2 Proximate composition, phosphorus content, and amino acid profile of the ingredients used in the growth and digestibility trials.

Proximate composition1 (As is g kg-1) Flash dried yeast Fish meal Soybean meal

Crude protein 387.8 627.8 448.9

Moisture 43 79.9 109.7

Crude fat 68.5 105.6 37.8

Crude fiber 62.7 0 32

Ash 36.6 187.5 66.7

Phosphorus 8.6 31.5 6.6

Amino acids profile (As is %)

Alanine 2.21 3.91 2.04

Arginine 2.00 3.68 3.35

Aspartic acid 3.39 5.34 5.10

Cysteine 0.43 0.47 0.62

Glutamic acid 4.36 7.47 8.24

Glycine 1.74 4.88 2.04

Histidine 0.94 1.63 1.20

Isoleucine 1.96 2.42 2.17

Leucine 3.43 4.21 3.57

Lysine 2.39 4.67 3.06

Methionine 0.80 1.61 0.66

Phenylalanine 1.94 2.39 2.35

Proline 1.89 3.08 2.39

Serine 1.77 2.11 1.90

Taurine 0.15 0.73 0.13

Threonine 1.83 2.41 1.75

Tryptophan 0.44 0.62 0.62

Tyrosine 1.48 1.67 1.64

Valine 2.20 2.99 2.34 1 Ingredients were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

31

Table 3 Proximate composition (g kg-1 as is) and amino acid profile (% as is) of the test diets used in the growth trial.

Proximate composition1 (As is g kg-1) D1 D2 D3 D4 D5

Crude protein 362.0 364.6 358.7 367.4 363.5

Moisture 79.2 74.1 85.2 67.7 68.2

Crude fat 79.7 94.6 85.9 95.8 103.8

Crude fiber 37.2 37.8 37.3 35.7 34.1

Ash 63.4 63.3 61.8 62.3 61.4

Amino acids profile (As is %)

Alanine 1.84 1.84 1.84 1.86 1.89

Arginine 2.19 2.2 2.19 2.19 2.21

Aspartic acid 3.34 3.33 3.32 3.32 3.35

Cysteine 0.49 0.49 0.50 0.50 0.50

Glutamic acid 7.12 7.03 6.94 6.97 7.02

Glycine 1.58 1.58 1.59 1.60 1.61

Histidine 0.88 0.88 0.88 0.89 0.90

Isoleucine 1.61 1.64 1.64 1.65 1.67

Leucine 3.19 3.20 3.18 3.21 3.25

Lysine 2.00 2.00 1.99 2.00 2.02

Methionine 0.60 0.60 0.60 0.61 0.62

Phenylalanine 1.82 1.82 1.81 1.82 1.84

Proline 2.36 2.32 2.31 2.33 2.34

Serine 1.67 1.59 1.58 1.58 1.64

Taurine 0.15 0.15 0.15 0.15 0.15

Threonine 1.31 1.30 1.30 1.31 1.34

Tryptophan 0.43 0.45 0.43 0.44 0.43

Tyrosine 1.26 1.27 1.27 1.28 1.29

Valine 1.77 1.80 1.80 1.82 1.83 1 Diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

32

Table 4 Composition of reference diet for the determination of digestibility coefficients of flash dried yeast (FDY), fishmeal (FM), and soybean meal (SBM).

Ingredients g kg-1 as is

Menhaden fish meal2 100.0

Soybean meal1 325.0

Menhaden fish oil2 32.0

Whole wheat3 476.0

Trace mineral premix4 5.0

Vitamin premix w/o choline5 18.0

Choline cloride5 2.0

Stay C6 1.0

Corn starch3 10.0

Lecithin7 10.0

Chromic oxide7 10.0 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Omega Protein Inc., Houston, TX, USA. 3 MP Biomedicals Inc., Solon, Ohio, USA. 4Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 5 Vitamin premix (g/kg premix): Thiamin.HCl, 4.95; Riboflavin, 3.83; Pyridoxine.HCl, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 6 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ,USA. 7 The Solae Company, St. Louis, MO, USA.

33

Table 5 Performance of juvenile Pacific white shrimp L. vannamei (1.78±0.03g) offered diets with different flash dried yeast (FDY) levels (0, 10, 20, 40, and 60 g kg-1) for six weeks.

Diet Final

biomass (g)

Final mean

weight (g) WG (%)4 FCR2 Survival (%) PRE (%)3

D1 103.04a 10.74a 503.84a 1.40b 96.0 36.93a

D2 105.03a 10.99a 516.94a 1.38b 96.0 37.32a

D3 102.70a 10.48a 498.64a 1.44b 98.0 37.08a

D4 97.16a 10.35a 489.64a 1.48b 94.0 35.44a

D5 86.38b 9.61b 441.09b 1.64a 90.0 32.52b

P-value 0.0007 0.0018 0.0076 <0.0001 0.4909 0.0124

PSE1 1.228 0.0928 5.986 0.0131 1.4422 0.9773 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 PRE: Protein retention efficiency = (final weight × final protein content) - (initial weight × initial protein content) × 100 / protein intake. 4 WG: Weight gain = (Final weight-initial weight)/initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

34

Table 6 Proximate analysis of whole shrimp body offered diets with different flash dried yeast (FDY) levels (0, 10, 20, 40, and 60 g kg-1) for six weeks.

Diet Proximate composition (g kg-1)

Protein2 Moisture Lipid2 Crude fiber2 Ash2

D1 810.9 776.4 65.3 56.1 120.2

D2 802.1 775.5 59.7 55.1 120.3

D3 807.3 773.0 59.8 56.9 124.7

D4 791.9 767.5 56.1 55.5 120.6

D5 807.7 771.8 50.0 52.8 119.9

P-value 0.2649 0.5580 0.0801 0.5548 0.7707

PSE1 2.8031 1.7898 1.6074 0.7855 1.3021 1 PSE: Pool standard error. 2 Dry weight basis.

35

Tab

le 7

App

aren

t dry

mat

ter

(AD

MD

), ap

pare

nt e

nerg

y (A

DE)

and

app

aren

t pro

tein

(A

DP)

dig

estib

ility

val

ues

for

the

diet

(D

) an

d in

gred

ient

(I) u

sing

70:

30 re

plac

emen

t tec

hniq

ue o

ffer

ed to

Pac

ific

whi

te s

hrim

p L.

van

nam

ei.

Die

t A

DM

DD

(%)

AD

ED (%

) A

DPD

(%)

AD

MD

I (%

) A

DEI

(%)

AD

PI (%

)

Bas

al d

iet 1

76

.38a

82.6

6a 92

.08a

Fl

ash

drie

d ye

ast

62.1

8c 69

.18c

79.4

7b 29

.04c

38.2

0b 53

.47c

Soyb

ean

mea

l 75

.42a

81.4

2a 94

.00a

73.1

8a 78

.20a

97.0

3a

Fish

mea

l 1

68.2

1b 78

.31ab

80

.86b

49.1

5b 69

.77a

67.0

7b

Bas

al d

iet 2

75

.93a

82.1

0a 92

.10a

Fish

mea

l 2

67.9

9b 76

.44b

82.3

4b 49

.45b

65.7

8a 71

.30b

PSE1

0.55

80

0.56

01

0.36

55

2.25

52

1.94

25

1.17

43

P-va

lue

<0.0

001

<0.0

001

<0.0

001

0.00

04

0.00

02

<0.0

001

1 PS

E: P

ool s

tand

ard

erro

r. FM

1 an

d FM

2 re

pres

ent t

he fi

rst c

olle

ctio

n an

d se

cond

col

lect

ion,

resp

ectiv

ely.

B

asal

die

t 1 a

nd B

asal

die

t 2 re

pres

ent f

irst c

olle

ctio

n an

d se

cond

col

lect

ion

basa

l die

t, re

spec

tivel

y.

Val

ues

with

in a

col

umn

with

diff

eren

t sup

ersc

ripts

are

sig

nific

antly

diff

eren

t bas

ed o

n Tu

key’

s m

ultip

le ra

nge

test

.

36

Table 8 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal (FM) and flash dried yeast (FDY) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei.

AA digestibility

coefficients (%) SBM FM FDY PSE1 P-value

Adjust

P-value

Alanine 93.75a 69.09b 51.31b 2.6712 0.0019 0.004

Arginine 96.91a 75.35b 67.83b 2.3361 0.0056 0.0069

Aspartic acid 95.39a 69.23b 62.40b 1.9463 0.0010 0.0029

Cysteine 91.29a 54.39b 34.80b 4.1270 0.0039 0.0055

Glutamic acid 95.69a 70.84b 56.83b 2.1174 0.0008 0.0028

Glycine 95.06a 66.55b 38.81c 3.1679 0.0011 0.0029

Histidine 94.33a 74.26ab 61.75b 2.9546 0.0115 0.0127

Hydroxylysine 97.28a 64.83b 39.89c 1.5132 <0.0001 <0.0001

Isoleucine 93.23a 68.72b 64.80b 2.1605 0.0034 0.0051

Leucine 92.23a 71.29b 60.02b 2.5186 0.0055 0.0069

Lysine 95.03a 76.97ab 66.88b 2.4289 0.0089 0.0104

Methionine 95.20a 70.63b 68.55b 1.4766 0.0006 0.0028

Phenylalanine 93.41a 65.28b 60.29b 2.4329 0.0029 0.0047

Proline 94.68a 67.21b 50.53b 2.8633 0.0022 0.0042

Serine 93.11a 58.31b 48.19b 2.4971 0.0008 0.0028

Taurine 50.56ab 90.28a 30.54b 6.0375 0.0180 0.0189

Threonine 91.99a 66.33b 59.45b 2.0842 0.0016 0.0037

Tryptophan 95.37a 80.31b 80.39b 0.6973 0.0002 0.0021

Tyrosine 95.28a 73.62b 72.85b 1.3263 0.0007 0.0028

Valine 90.78a 67.06ab 59.88b 3.5325 0.0271 0.0271

Total AA 94.31a 69.91b 58.33b 2.4113 0.0024 0.0042 1 Pooled standard error Values within a row with different superscripts are significantly different based on Tukey’s multiple range test.

37

CHAPTER III

EVALUATION OF THREE NON-GENETICALLY MODIFIED SOYBEAN

CULTIVARS AS INGREDIENTS AND A YEAST-BASED ADDITIVE AS A

SUPPLEMENT IN PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus

vannamei

1. Introduction

Soybean meal (SBM) is usually considered as the most reliable ingredient and cost-

effective protein source in shrimp feed because of its worldwide availability, low price, relatively

balanced amino acid profile, and consistent composition (Amaya et al. 2007b; Davis & Arnold

2000). There have been a number of studies demonstrating the successful use of conventional

SBM in practical diets for Pacific white shrimp, Litopenaeus vannamei (Amaya et al. 2007b;

Roy et al. 2009; Sookying & Davis 2011; Zhu et al. 2013). Albeit commodity SBM is still an

acceptable protein source with good digestibility for shrimp, it also has some potential

limitations associated with insufficient levels of essential amino acids (EAA) such as methionine

and lysine, presence of anti-nutritional factors (ANFs) for example trypsin inhibitors, and poor

palatability, which may limit its potential use in feed formulations (Dersjant-Li 2002).

Traditionally, supplementation methionine and lysine and inclusion of attractants or palatability

enhancers in SBM-based diets to meet the shrimp requirement is recommended to provide a

good growth response (Akiyama 1989). Also, ANFs such as trypsin inhibitors present in raw

soybean can negatively affect nutrient digestion and availability but this can be reduced or

eliminated through heat processing (Dersjant-Li 2002; New 1987).

38

Alternatively, novel soy breeding technology can be used to develop new soybean

cultivars, which selectively decrease certain heat-resistant ANFs (such as oligosaccharides),

while increasing the protein content of the resulting meals. Building on this progress,

unconventional, non-thermal (energy-saving), nutrient-preserving processing methods can be

used to produce these novel meals. The resulting meals are of considerable interest in

aquaculture because they may potentially provide improved feed ingredients with no need for

genetic modifications of the native soy DNA, therefore, resulting in nutritionally superior

products (Fang et al. 2016). In addition, selective soy breeding allows for a tight control on some

functional compounds that may promote immunogenicity in shrimp. As shrimp culture has

become an expanded and intensified economic activity, bacterial and viral diseases are

considered a threat to the future progress of semi-intensive and intensive shrimp culture (Pérez‐

Sánchez et al. 2014). Traditional antibiotics supplementation can prevent or treat diseases but it

may also develop resistant bacterial strains immune to antibiotic treatment. In addition, the

antibiotic residues in cultured animals may act in detriment of human health (Cabello 2006;

Sharifuzzaman & Austin 2009; Taylor et al. 2011). To tackle disease problems and avoid

potential disadvantages of antibiotic use, alternative strategies, for example the use of probiotics

(e.g., yeast) and prebiotics has received considerable attention in recent years (Li & Gatlin 2005).

Products of various fermented yeast, not containing live/viable cells, often contain

primary metabolites, such as nucleotides, polysaccharides, small peptides, organic acids and

lipids. Some of these materials have been demonstrated to improve the growth, survival, and

immune response of L. vannamei in several studies (Deng et al. 2013; Tipsemongkol et al. 2009).

Our laboratory has already evaluated digestibility values of some new non-genetically

modified (non-GM) soybean cultivars and the effects on the growth performance of L.vannamei

39

(Fang et al. 2016; Zhou et al. 2015). However, information about the impacts of novel

processing on selected soybean cultivars on immune response is still needed. Therefore, the

purposes of this study were to investigate the effects of three new, non-GM soybean cultivars

processed with novel technologies on the growth performance and immune responses of L.

vannamei and to verify the effects of a fermented yeast product on growth and immunity of L.

vannamei.

2. Materials and Methods

2.1 Ingredients preparation

Four sources of SBM, including three new, non-GM soybean cultivars, were obtained for

evaluation of their potential use as ingredients in L. vannamei feeds. Commodity SBM soy was

obtained from Bunge Limited, Decatur, AL, USA. The non-GM ingredients were processed via

proprietary technology and donated by NavitaTM Premium Feed Ingredients (NPFI), West Des

Moines, IA, USA. They were analyzed at University of Missouri Agricultural Experiment

Station Chemical Laboratories (Columbia, MO, USA) for proximate composition, phosphorus

content, trypsin inhibitor, urease activity, acid detergent fiber (ADF), neutral detergent fiber

(NDF), protein dispersibility index (PDI), starch, and amino acid profiles in the diets were

formulated (Tables 2).

2.2 Experiment design and diets

Two growth trials were conducted to evaluate biological response of shrimp to three

novel soybean cultivars and a fermented yeast commercial product in practical diets with regards

40

to growth and feed utilization. All test diets were formulated to be iso-nitrogenous and iso-lipidic

(35% protein and 8% lipid). A total of 9 experimental diets were formulated (Table 1). The basal

diet (1) was primarily composed of a commodity SBM, fishmeal (FM), whole wheat, corn

protein concentrate, poultry by-product meal (PM, pet food grade) and corn starch. For the

experimental diets, either low (L) or high (H) inclusion levels of three novel soybean ingredients

(N1-N3) were utilized to replace all the conventional SBM and to partially substitute FM and

PM in the basal diet. Additionally, the last two diets (8 and 9) were essentially the same as diets

1 and 3 but supplemented with a commercially available fermented yeast product.

Pre-ground dry ingredient and oil were mixed in a food mixer (Hobart Corporation, Troy,

OH, USA) for 15 minutes. Hot water was then blended into the mixture to obtain a consistency

appropriate for pelleting. Diets were pressure-pelleted using a meat grinder with a 3 mm die, air-

dried (<50 °C) to a moisture content of 8-10%. Pellets were crumbled, packed in sealed plastic

bags and stored in a freezer (-20 °C) until needed. As the ingredients, the diets were also

analyzed at University of Missouri for proximate composition (g 100 g-1 as is) and amino acid

profile (g 100 g-1 as is) (Table 3).

2.3 Growth trials

Two trials were conducted at the E.W. Shell Fisheries Research Station, Auburn

University (Auburn, AL, USA). Pacific white shrimp post larvae (PL) were obtained from

Shrimp Improvement Systems (Islamorada, Florida) and nursed in an indoor recirculating

system. PLs were fed a commercial feed (Zeigler Bros., Inc., Gardners, Pennsylvania, USA)

using an automatic feeder for ~1 week, and then switched to crumbled commercial shrimp feed

(Rangen® Inc., Buhl, Idaho, USA) for ~1- 2 weeks.

41

At the end of the nursery phase, juvenile shrimp (initial mean weight 0.15 g for trial 1;

0.20 g for trial 2) were obtained from the nursery system and hand-sorted to uniform size. Both

growth trials utilized 9 treatments with 6 replicates in each treatment. In trial 1, juvenile shrimp

were stocked into 54 tanks with 15 shrimp per aquarium in a semi-recirculation system. In trial

2, juvenile shrimp were stocked into 54 tanks with 10 shrimp per aquarium in the same semi-

recirculation system as the trial 1. The semi-recirculation system consisted of 54 aquaria (60 L)

connected to a common reservoir, biological filter, bead filter, fluidized biological filter and

recirculation pump.

During the two trials, shrimp were fed four times daily over 46 days for trial 1 and 35

days for trial 2. Daily allowances of feed were adjusted based on observed feed consumption,

weekly counts of the shrimp and mortality. Based on the historic results in our laboratory, daily

feed rations were initially calculated assuming a 1.8 FCR and doubling in size the first three

weeks for trial 1, first two weeks for trial 2 and an increment of 0.8 g/week thereafter.

Consequently, for each tank in trial 1, a fixed ration of 0.58 g day-1 for the first week, 1.16 g day-

1 for the second week, 2.31 g day-1 for the third week, and 3.09 g day-1 for the remaining

culturing period was offered, partitioned in 4 feedings each day. For each tank in trial 2, a fixed

ration of 0.51 g day-1 for the first week, 1.03 g day-1 for the second week, 1.85 g day-1 for the

third, forth, and fifth week was also allotted in 4 portions per day. At the conclusion of each

growth trial, shrimp were counted and group-weighted. Mean final weight, FCR, WG, biomass,

and survival were determined (Table 4).

42

2.4 Physiological assessment

A second recirculation system was used to condition sub-adult shrimp for the collection

of physiological samples. The culture system was equivalent to the previously described system

but utilized a series of 130 L aquaria which were stocked with 6 sub-adult shrimp (14.8 g mean

weight). Shrimp were offered diets 1, 3, 8, and 9 to slight excess over a 15 days period. At the

conclusion of the conditioning period the shrimp were sacrificed and hemolymph samples were

collected for assessment of total hemocyte count (THC), hyaline cells count (HCC), granular

cells count (GCC), semi-granular cells count (SGCC), hemolymph glucose (HG), hemolymph

packed cells volume (HPCV), hemolymph protein (HP), hemocyte phagocytic capacity (HPC),

and hemocyte respiratory burst activity (HRBA) (Table 8). A sample of hemolymph was

obtained from each shrimp using a 25 gauge needle through the dorsal surface, and the glucose

level was determined using a handheld glucometer (Abbott Diabetic Care, Inc., Alameda, CA).

The remainder of each sample was loaded into a hematocrit tube, capped and centrifuged at

10,000 rpm for 5 minutes, and the packed hemocyte volume was read using a Micro-Hematocrit

Capillary Tube Reader (Monoject Scientific, St. Louis, MO). After this step, the hemolymph

protein content was determined by placing a drop of the supernatant onto a handheld protein

refractometer (VEEGEE Scientific Inc. Kirkland, WA). An additional sample of hemolymph was

collected into an equal volume of anticoagulant and a 20 µl aliquot of the hemolymph and

anticoagulant mixture was added to an equal volume of Trypan Blue and allowed to sit for 1

minute. Cells were counted using a hemocytometer, and one corner of 16 squares was read. Cell

number per ml for both total and differential counts was determined by the following formula:

cell number x dilution factor (4) x 106. After cell counting was completed, the cell numbers were

standardized using PBS to dilute the samples. The respiratory burst activity of the cells was

43

assessed in the following manner using the standardized samples: 100 µl of sample and 100 µl of

zymosan solution were added in triplicate to a 96-well plate and incubated at room temperature

for 30 minutes. The zymosan solution was then removed, the wells were rinsed three times with

100 µl PBS, and 100 µl of NBT solution was added and incubated for 30 minutes and removed.

The wells were then fixed with absolute methanol, and rinsed three times with 70% methanol.

Subsequently, 120 µl of KOH and 140 µl DMSO were added to dissolve the DBT diforazon

precipitate. Plates were then read in a spectrophotometer at 600 nm for comparison of

absorbance (Yeh et al. 2004). To determine phagocytic capacity, an additional sample of 50 µl of

hemolymph with anticoagulant was loaded induplicate on Esco Fluor glass slides and incubated

at room temperature for 90 minutes. After incubation, 50 µl of zymosan solution was added and

incubated at room temperature for 60 minutes. The slides were then washed with PBS, air dryed,

and fixed in methanol, and stained with Wright stain. At least 100 hemocytes per slide were

microscopically examined at 100 x magnification for evidence of phagocytic activity, and

phagocytic capacity was determined by the percentage containing 5 or more zymosan particles

(Mustafa et al. 2000).

2.5 Water quality monitoring

For both trials, dissolved oxygen (DO), water temperature and salinity were measured

twice daily by using a YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA).

Hydrogen potential (pH) was measured twice weekly by using a waterproof pHTestr30 (Oakton

instrument, Vernon Hills, IL, USA). Total ammonia-nitrogen (TAN) and nitrite were evaluated

every week by using the methods described by Sororzano (1969) and Spotte (1979).

44

2.6 Statistical analysis

All data was analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). For the growth

trial data was analyzed using one-way ANOVA to determine significant differences (P < 0.05)

among treatments followed by Tukey’s multiple comparison test to determine difference

between treatment means. Principle component analysis (PCA) was performed to reduce the

dimensions. Multiple linear regression was utilized to detect the relationship of WG or FCR with

principle components selected from PCA. Correlation coefficient analysis was utilized to identify

the relationships between trypsin inhibitor, methionine and lysine levels in the diets and the

shrimp’s biological responses. Data from the physiological assessment was analyzed using two-

way ANOVA to evaluate soy sources across fermented yeast product supplementation levels.

3. Results

3.1 Water quality

In trial 1, DO, temperature, salinity, pH, TAN, and nitrite were maintained at

6.01±0.38mg L-1, 27.8±1.1°C, 7.4±0.3ppt, 7.21±0.20, 0.068±0.037mg L-1, and 0.201±0.090mg

L-1, respectively. In trial 2, DO, temperature, salinity, pH, TAN, and nitrite were maintained at

5.63±0.28mg L-1, 29.3±0.4°C, 5.1±0.1ppt, 7.67±0.22, 0.030±0.023mg L-1, and 0.023±0.016mg

L-1, respectively. Water quality conditions in both of the trials were suitable for normal growth

and survival of this species.

3.2 Growth trials

Growth performance of juvenile L. vannamei offered diets with different ingredients are

presented in Table 4. In trial 1, significant differences were detected in final biomass, final mean

45

weight, WG, FCR, and survival when shrimp were fed with various diets. Highest final biomass

(65.74 g) was observed in shrimp fed with the basal diet. Shrimp fed diets 2 and 3 (LN1 and

HN1, respectively) exhibited the highest final mean weight (5.08 g) and WG (3,463%), while

shrimp fed diet 5 (HN2) had the lowest values for those two indicators (4.06 g and 2,627%,

respectively). The FCR range spanned from 1.38 to 1.75 and the lowest value was observed in

shrimp fed diet 2 (LN1), while the highest FCR was found in shrimp fed diet 3 (HN2). Survival

ranged from 65.6 to 91.1% and the lowest survival was observed in shrimp fed diets 2 and 3. In

contrast, the highest survival was documented for shrimp fed the basal diet.

Similarly, trial 2 revealed that final biomass, final mean weight, WG, and FCR were

significantly affected by dietary treatment. Shrimp fed diet 2 had the highest final biomass, final

mean weight, and WG (36.72 g, 4.02 g, and 2,050%, respectively), while shrimp fed diet 3

exhibited the lowest values for these indices (27.32 g, 3.29 g, 1,519%, respectively). In trial 2

FCR ranged from 1.34 to 1.74 and the lowest value was observed in shrimp fed diet 8, while the

highest FCR was found in animals fed diet 3. In this second trial survival ranged from 78.3 to

93.3% but no significant differences were observed among shrimp fed the various diets.

Principle component analysis (PCA) of trypsin inhibitors and essential amino acids and

their loadings of experimental diets are presented in Table 5. The first principle component (PC)

explained 71.1% of total sample variance. Collectively, the first three PCs explained 92.86% of

total samples variance. Loadings of essential amino acids are similar in PC1 (ranged from 0.234

to 0.347). The only negative loading in PC1 is trypsin inhibitor (-0.153). In PC2, loadings of

methionine and lysine are -0.495 and -0.205 respectively. Trypsin inhibitor (loading=0.836) is in

charge of the third PC.

46

The results of multiple linear regression of WG and FCR on the first three PCs (PC1,

PC2, and PC3) are presented in Table 6. P-value for all the models are less than 0.05. In trial 1,

PC3 has a significantly negative impact on WG while it has positive effect on FCR. In trial 2,

PC2 and PC3 have significantly negative influence on WG, however they have positive effect on

FCR. The first PC has significantly positive effect on WG while it has negative effect on FCR.

Combined the results of PCA and multiple linear regression, we may conclude that the

significantly reduced growth may be attributed to the high trypsin inhibitor level and relatively

insufficient essential amino acids (mainly methionine and lysine) levels as well as their

combined effects.

Pearson correlation coefficients of growth performances (final biomass, final mean

weight, and WG), FCR and survival with dietary trypsin inhibitor, methionine or protein levels

are presented in Table 7. In trial 1 and 2, dietary trypsin inhibitor levels negatively correlated

with growth performance, while positively correlating with FCR. Similarly, dietary lysine

positively correlated with WG while negatively correlating with FCR. Finally, methionine levels

positively correlated with WG while negatively correlating with FCR in trial 2. However, no

correlations of methionine levels were found for WG or FCR in trial 1.

3.3 Physiology trial

The two-way ANOVA analysis for stress and immune responses of L. vannamei fed

different soy ingredients and fermented yeast are presented in Table 8. The combined effect

soy*yeast was significant (P = 0.035) for hemolymph glucose (HG) but the most significant

effect (P = 0.003) was found for the fermented yeast significantly reducing granular cells counts

(GCC). Another important effect was identified when soy ingredients significantly reduced the

47

hemolymph pack cells volume (HPCV). No significant differences were observed in total

hemocyte counts (THC), hyaline cells counts (HCC), semi-granular cells counts (SGCC),

hemolymph protein (HP), hemocyte phagocytic capacity (HPC), or hemocyte respiratory burst

activity (HPBA).

4. Discussion

Dietary protein is one of the most important factors affecting growth performance of

shrimp and feed cost (Hu et al. 2008). Reducing or replacing costly animal protein sources

through the use of more economical plant protein sources such as SBM could result in

substantial saving in feed cost. However, the presence of ANFs in SBM may affect the digestion

and reduce nutrient availability to shrimp (Dersjant-Li 2002), thus limiting the inclusion level of

SBM in practical shrimp diets. By using novel soybean breeding technology, improved soybean

varieties with reduced levels of ANFs and enhanced protein and lipid content could improve

nutrient bioavailability, and therefore allow for the supplementation of these ingredients in

practical shrimp feeds at higher concentrations preventing adverse effects on the digestive

physiology of shrimp. An added benefit of these novel ingredients, compared to commodity

SBM is that these typically have higher protein content which could help meet the shrimp’s

dietary protein requirement at a lower inclusion level; thus, opening more formulation space in

the diet (Fang et al. 2016).

SBM has been successfully utilized as a protein source in practical diets for Pacific white

shrimp. Amaya et al. (2007b) demonstrated that FM could be completely replaced with plant

protein by including 39.52% SBM in combination with 16% PM in practical shrimp feeds

without compromising production or economic performance of L. vannamei reared in ponds. In a

48

different study, Amaya et al. (2007a) also confirmed that good performance can be attained when

shrimp is fed a plant protein-based feed with 56.46% solvent extracted SBM in combination with

1% squid meal (SM). Moreover, SBM levels up to 58% have been demonstrated to be feasible in

practical shrimp diets without adversely affecting WG, survival, or FCR (Roy et al. 2009;

Sookying & Davis 2011). The apparent success of such high dietary SBM levels indicates that

this product can serve as the primary protein source in practical shrimp diets and thus, the further

evaluation of novel soy cultivars and processing technologies is paramount to further enhance

shrimp aquaculture.

Protein quality of SBM is linked to both the reduction of ANFs and the optimization of

protein digestibility. Of tests commonly used, the evaluation of urease activity (UI) and trypsin

inhibitors are especially useful to identify under processed SBM (Căpriţă et al. 2010). PDI

measures the dispersibility of proteins in water and is generally used as qualitative parameter of

protein denaturation upon thermal processing of SBM (Qin et al. 1996). Manufacturers of soy-

based fish and shrimp feeds require low PDI values to prevent losses of valuable protein into the

aquatic environment (Reinitz 1984). Combining results from PDI and urease tests could become

a useful tool to monitor SBM quality. In the current study, protein contents of ingredients N1-3

(55.33, 47.61, and 49.38 % versus 45.98 %) and lipid contents of N2 and N3 were enhanced

(4.98 and 6.66 % versus 1.54 %) compared to the commodity SBM by using the novel soy

breeding technology (Table 2). However, the trypsin inhibitor levels, urease activity, and PDI of

N2 and N3 were also higher than the commodity SBM (18,423 and 16,943 TIU g-1 vs 8,656 TIU

g-1, 2.27 and 2.20 vs 0.04, and 49.17 and 85.19 vs 26.69) (Table 2). This is due to the fact that

N1 originates in a different cultivar than N2 and N3 and although the second cultivar has

significantly less trypsin inhibitors, the novel processing options utilized were not able to

49

sufficiently reduce these indices to levels comparable to traditional roasting of SBM. Because as

oil is extracted a concomitant concentration of protein occurs in soy products and due to the fact

that both the Kunitz and Bowman-Birk protease inhibitors are proteins, more research is needed

to fine tune the non-thermal novel processing options such that these are able to attain similar

levels of ANFs inactivation as traditional heat treatments (DiPietro & Liener 1989). The

suboptimal inactivation resulted in higher trypsin inhibitors levels in the diets (Table 4).

Trypsin inhibitors have been demonstrated to result in growth depression and pancreatic

hypertrophy in numerous experimental animals (Lim & Akiyama 1992), such as Atlantic salmon

Salmo salar (Olli et al. 1994) and Rainbow trout Oncorhynchus mykiss (Kaushik et al. 1995) as

well as Pacific white shrimp (Fang et al. 2016; Zhou et al. 2015). Negative effects on protein

digestibility have been attributed to trypsin inhibitors due to their capacity to bind digestive

enzymes (Francis et al. 2001), which may negatively affect growth performance of the Pacific

white shrimp. In the present study, shrimp fed diet 5 (HN2) showed significantly reduced WG

compared to the shrimp fed diets 2 (LN1) in trial 1 and 2, as well as compared to shrimp fed with

diets 1, 3 and 8 in trial 2. The FCR was significantly increased when shrimp consumed diet 3

(HN2) in contrast with shrimp fed with diets 1, 2, 3, and 8 in trial 1 and 2 as well as compared to

shrimp fed diet 6 (LN3) in trial 2. The trends of the WG and FCR in trial 1 were similar to those

in trial 2, which points to consistency between results of the two trials. There were slight

differences in the statistics analysis results, which may have resulted from data variation.

In both of the trials, shrimp fed diet 5 exhibited lowest growth across all the treatments.

As diet 5 contained highest trypsin inhibitor level and lowest methionine and lysine levels, we

suspect if trypsin inhibitor level and methionine and lysine levels caused the depressed growth.

In order to demonstrate our thoughts, PCA and multiple regression analysis were performed. In

50

trial 1, the multiple regression results showed that PC3 had a significantly negative impact on

WG while it had positive effect on FCR. From the loading of PC3 (Table 5), PC3 is positively

dominated by trypsin inhibitor level (loading=0.836). Combined the results of PCA and multiple

regression analysis in trial 1, we can infer that trypsin inhibitor pose negative effect on WG but

positive effect on FCR. In trial 2, PC2 and PC3 have significantly negative influence on WG,

however they have positive effect on FCR. PC2 is negatively dominated by lysine (loading=-

0.495) followed by methionine (-0.205). Combined the results of PCA and multiple regression

analysis in trial 2, we can infer that trypsin inhibitor pose negative effect on WG but positive

effect on FCR. Methionine and lysine levels had positive effect on WG but negative effect on

FCR. Therefore, from the results of PCA and multiple regression analysis, we may infer that the

significantly reduced growth may be attributed to the high trypsin inhibitor level and relatively

insufficient essential amino acids (typically methionine and lysine) levels as well as their

combined effects. Also, the results of correlation analysis in the present experiment confirmed

that protease inhibitor levels in the diets negatively correlated with shrimp growth while

positively correlated with FCR. Similarly, Zhou et al. (2015) and Fang et al. (2016) reported that

the growth performance of Pacific white shrimp was significantly improved when shrimp fed

with diets contained different soybean cultivars with a reduced level of trypsin inhibitors. Based

on correlation analysis, trypsin inhibitors levels in the soybean meal cultivars were confirmed to

be negatively correlated with protein digestibility (Zhou et al. 2015) and shrimp growth (Fang et

al. 2016).

Methionine and lysine are generally the most limiting amino acids in the plants and

rendered animal byproducts. The correlation analysis in the current study demonstrated that

lysine level in the diets positively correlates with growth performance while negatively

51

correlating with FCR in both trial 1 and 2. No correlation of methionine with WG and FCR were

observed in trial 1, while methionine positively correlated with WG while negatively correlated

with FCR in trial 2. The dietary lysine requirement for the Pacific white shrimp estimated by the

broken-line model based on specific growth rate (SGR) was 2.05% of diet (dry weight basis) or

4.93% of dietary protein (Xie et al. 2012). With regards to the methionine requirement, the

information for Pacific white shrimp is still limited. In the present study, dietary lysine and

methionine levels (Table 4) were both lowest for diets 5 (lysine: 1.89% of diet, methionine:

0.51% of the diet) which might partially explained relatively reduced WG and increased FCR in

this treatment.

In the current study, fermented yeast supplementation at 0.13% of diet did not affect the

WG of the shrimp. Similarly, Tipsemongkol et al. (2009) reported that 0.125% supplementation

of a fermented yeast product did not affect the WG of Pacific white shrimp. However, WG of

shrimp was significantly improved when the supplementation increased to 0.25%. By contrast,

Deng et al. (2013) indicated that final mean weight of Pacific white shrimp was significantly

improved under pond conditions when a fermented yeast product was included at both 1.0 and

1.5 g kg-1. In pond trials, natural food is available to shrimp, which may probably mask the

effects of fermented yeast products on the growth performance of shrimp, thus causing the

differences among these studies.

As for the physiological trial, independent and combined effects of the fermented yeast

product and the soy ingredients were evaluated in a 2 × 2 factorial design (Table 7). An elevation

of hemolymph glucose (HG) is considered as indicator of stress responses in invertebrates

(Chang et al. 2016). Hemolymph packed cells volume (HPCV) measures the volume percentage

of red blood cells in vertebrates and hemocytes in shrimp. A higher HPCV level is indicative of

52

an increased capacity of the hemocoel to transport oxygen (Birchard 1997). In the current study,

the shrimp fed diets 3, 8 and 9 (HN1, and yeast product) exhibited a significantly higher HG and

lower HPCV content, which indicates that the shrimp in these treatments were stressed. No other

independent or combined effects of these particular diets were observed on immune responses

(THC, HC, SGC, and HPC) of shrimp. However, GC in shrimp fed diets containing the yeast

product was significantly lower than those fed yeast-free diets. Since no decreasing trends were

observed in other immune responses indicators, the reduction of GC in the current study might

be odd or due to the testing errors. Similarly, Tipsemongkol et al. (2009), reported that immune

responses (THC, percentage phagocytosis, superoxide dismutase, and phenol oxidase activity) of

Pacific white shrimp were not affected after 10, 20, and 30 days of feeding with a fermented

yeast product at 0.125%. However, in the same study 0.125% supplementation of a fermented

yeast product in shrimp diets significantly enhanced immune responses after 40 and 50 days of

feeding with the product at 0.125% (Tipsemongkol et al. 2009). The time frame for the present

physiological evaluation was only 15 days, which may be insufficient for immune system of

shrimp to develop detectable responses to the inclusion of dietary fermented yeast.

5. Conclusion

In the present study, the growth performance of shrimp fed N1 diets was highest among

the novel soy ingredients and the commodity SBM. This indicates that breeding technology and

novel soy processing has the potential to increase the nutritional values of SBM for shrimp feeds.

Observed trends on immune indicators of shrimp to both independent and combined effects of

soy ingredients and fermented yeast were not easily discernible and may be related to a

suboptimal exposure period.

53

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57

Tab

le 1

For

mul

atio

n an

d ch

emic

al c

ompo

sitio

n of

test

die

ts u

sed

in th

e gr

owth

tria

l

Ingr

edie

nt (

As i

s bas

is g

kg-1

feed

)

Die

t cod

e

1 2

3 4

5 6

7 8

9

Bas

al

LN1

HN

1 LN

2 H

N2

LN3

HN

3 B

asal

+yea

st

HN

1+ye

ast

Fish

mea

l1 12

0.0

120.

0 60

.0

120.

0 60

.0

120.

0 60

.0

120.

0 60

.0

Poul

try m

eal5

20.0

20

.0

5.0

20.0

5.

0 20

.0

5.0

20.0

5.

0

Soyb

ean

mea

l2 43

4.0

- -

- -

- -

434.

0 -

Nav

ita 1

6 -

322.

0 41

6.0

- -

- -

- 41

6.0

Nav

ita 2

6 -

- -

367.

0 47

4.0

- -

- -

Nav

ita 3

6 -

- -

- -

372.

0 48

0.0

- -

Cor

n pr

otei

n co

ncen

trate

3 20

.0

20.0

8.

0 20

.0

8.0

20.0

8.

0 20

.0

8.0

Who

le w

heat

4 30

0.0

300.

0 30

0.0

300.

0 30

0.0

300.

0 30

0.0

300.

0 30

0.0

Ferm

ente

d ye

ast p

rodu

ct

- -

- -

- -

- 1.

3 1.

3

Men

hade

n fis

h oi

l2 48

.7

52.6

60

.1

38.0

41

.3

28.2

28

.5

48.7

60

.1

Trac

e m

iner

al p

rem

ix7

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

Vita

min

pre

mix

8 18

.0

18.0

18

.0

18.0

18

.0

18.0

18

.0

18.0

18

.0

Cho

line

chlo

ride4

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

Stay

C9

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Mon

o-di

calc

ium

pho

spha

te10

19

.0

19.0

25

.0

19.0

25

.0

19.0

25

.0

19.0

25

.0

Leci

thin

11

10.0

10

.0

10.0

10

.0

10.0

10

.0

10.0

10

.0

10.0

Cho

lest

erol

4 0.

5 0.

5 0.

5 0.

5 0.

5 0.

5 0.

5 0.

5 0.

5

Cor

n st

arch

4 1.

8 10

9.9

89.4

79

.5

50.2

84

.3

57.0

0.

5 88

.1

58

1 O

meg

a Pr

otei

n In

c., H

oust

on T

X, U

SA.

2 D

e-hu

lled

solv

ent e

xtra

ct so

ybea

n m

eal,

Bun

ge L

imite

d, D

ecat

ur, A

L, U

SA.

3 Em

pyre

al®

75,

Car

gill

Cor

n M

illin

g, C

argi

ll, In

c., B

lair,

NE,

USA

. 4 M

P B

iom

edic

als I

nc.,

Solo

n, O

hio,

USA

. 5 T

yson

Foo

ds, I

nc.,

Sprin

gdal

e, A

R, U

SA.

6 N

avita

Pre

miu

m F

eed

Ingr

edie

nts,W

est D

es M

oine

s, IA

, USA

.

7 Tr

ace

min

eral

pre

mix

(g/

100

g pr

emix

): C

obal

t ch

lorid

e, 0

.004

; C

upric

sul

fate

pen

tahy

drat

e, 0

.550

; Fe

rrou

s su

lfate

, 2.

000;

M

agne

sium

sulfa

te a

nhyd

rous

, 13.

862;

Man

gane

se su

lfate

mon

ohyd

rate

, 0.6

50; P

otas

sium

iodi

de, 0

.067

; Sod

ium

sele

nite

, 0.0

10; Z

inc

sulfa

te h

epta

hydr

ate,

13.

193;

Alp

ha-c

ellu

lose

, 69.

664.

8 V

itam

in p

rem

ix (g

/kg

prem

ix):

Thia

min

.HC

L, 4

.95;

Rib

ofla

vin,

3.8

3; P

yrid

oxin

e.H

CL,

4.0

0; C

a-Pa

ntot

hena

te, 1

0.00

; Nic

otin

ic a

cid,

10

.00;

Bio

tin, 0

.50;

fol

ic a

cid,

4.0

0; C

yano

coba

lam

in, 0

.05;

Ino

sito

l, 25

.00;

Vita

min

A a

ceta

te (

500,

000

IU/g

), 0.

32;

Vita

min

D3

(1,0

00,0

00 IU

/g),

80.0

0; M

enad

ione

, 0.5

0; A

lpha

-cel

lulo

se, 8

56.8

1.

9 St

ay C

®, (

L-as

corb

yl-2

-pol

ypho

spha

te 2

5% A

ctiv

e C

), D

SM N

utrit

iona

l Pro

duct

s., P

arsi

ppan

y, N

J, U

SA.

10 J.

T. B

aker

®, M

allin

ckro

dt B

aker

, Inc

., Ph

illip

sbur

g, N

J, U

SA.

11 Th

e So

lae

Com

pany

, St.

Loui

s, M

O, U

SA.

59

Table 2 Proximate analysis (as is g 100 g-1), trypsin inhibitors levels (TIU g-1), acid detergent fiber (ADF) and neutral detergent fiber (NDF) (as is g 100 g-1), phosphorus (as is g 100 g-1), urease activity (pH increase), protein dispersibility index (PDI), starch (as is g 100 g-1) and amino acid (AA) profiles (as is g 100 g-1) of solvent extracted soybean meal (SBM), navita 1, 2, and 3 utilized in the trials.

Composition1 Solvent extracted SBM Navita 1 Navita 2 Navita 3

Crude Protein 45.98 55.33 47.61 49.38

Moisture 13.61 9.94 11.35 7.49

Crude Fat 1.54 0.58 4.98 6.66

Crude Fiber 3.18 4.44 4.46 4.78

Ash 5.73 6.28 5.64 5.91

ADF 6.93 14.9 10.86 15.09

NDF 7.24 13.87 14.31 10.82

Phosphorus 0.63 0.75 0.56 0.58

Trypsin inhibitor 8656 9396 18423 16943

Urease activity 0.04 0.06 2.27 2.20

PDI 26.69 15.22 49.17 85.19

Starch 2.23 0.00 0.74 0.14

Alanine 1.88 2.33 2 2.08

Arginine 3.18 4.1 3.55 3.64

Aspartic Acid 4.84 6.24 5.33 5.41

Cysteine 0.60 0.76 0.64 0.65

Glutamic Acid 7.51 9.66 8.34 8.51

Glycine 1.83 2.29 1.98 2.03

Histidine 1.27 1.44 1.24 1.25

Hydroxylysine 0.15 0.05 0.18 0.08

Hydroxyproline 0.08 0.06 0.06 0.06

Isoleucine 2.06 2.54 2.17 2.21

Leucine 3.56 4.26 3.67 3.77

Lysine 2.89 3.49 2.93 3.06

Methionine 0.59 0.75 0.65 0.65

Ornithine 0.07 0.06 0.04 0.05

60

Phenylalanine 2.34 2.78 2.42 2.46

Proline 2.30 2.75 2.36 2.41

Serine 1.92 2.42 2.13 2.15

Taurine 0.12 0.08 0.08 0.07

Threonine 1.70 2.1 1.82 1.86

Tryptophan 0.75 0.73 0.58 0.62

Tyrosine 1.85 1.86 1.78 1.81

Valine 2.05 2.69 2.3 2.36 1 Ingredients were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory.

61

Tab

le 3

Pro

xim

ate

anal

ysis

(as i

s g 1

00 g

-1),

tryps

in in

hibi

tors

leve

ls (T

IU/g

), an

d am

ino

acid

(AA

) pro

files

(as i

s g 1

00 g

-1) o

f the

test

di

ets u

tiliz

ed in

the

trial

s.

Com

posi

tion1 (A

s is %

)

Die

t cod

e

1 2

3 4

5 6

7 8

9

Bas

al

LN1

HN

1 LN

2 H

N2

LN3

HN

3 B

asal

+yea

st

HN

1+ye

ast

Cru

de P

rote

in

35.4

2 34

.17

32.7

3 34

.20

34.0

2 33

.54

33.7

0 35

.51

32.9

1

Moi

stur

e 9.

83

6.61

9.

95

6.46

6.

28

9.30

7.

71

9.04

9.

46

Cru

de F

at

8.42

9.

17

7.82

8.

72

8.72

7.

80

7.61

8.

74

8.32

Cru

de F

iber

3.

45

2.93

3.

23

4.22

4.

09

3.31

3.

58

3.43

3.

12

Ash

7.

59

7.19

6.

68

7.19

6.

92

6.93

6.

86

7.48

6.

70

Tryp

sin

Inhi

bito

r 22

44

2185

21

89

7358

81

12

6848

79

53

3207

21

89

Ala

nine

1.

67

1.60

1.

44

1.61

1.

45

1.54

1.

46

1.65

1.

42

Arg

inin

e 2.

10

2.07

2.

13

2.04

2.

16

2.03

2.

11

2.17

2.

12

Asp

artic

Aci

d 3.

15

3.08

3.

12

3.02

3.

16

2.93

3.

13

3.22

3.

12

Cys

tein

e 0.

43

0.43

0.

43

0.42

0.

44

0.40

0.

42

0.45

0.

44

Glu

tam

ic A

cid

6.33

6.

20

6.22

6.

10

6.28

5.

92

6.28

6.

35

6.16

Gly

cine

1.

79

1.66

1.

46

1.72

1.

48

1.59

1.

48

1.71

1.

46

His

tidin

e 0.

93

0.91

0.

89

0.90

0.

89

0.87

0.

88

0.93

0.

89

Hyd

roxy

lysi

ne

0.09

0.

08

0.07

0.

11

0.12

0.

09

0.07

0.

08

0.07

Hyd

roxy

prol

ine

0.25

0.

17

0.10

0.

22

0.10

0.

17

0.11

0.

16

0.10

Isol

euci

ne

1.54

1.

49

1.45

1.

49

1.48

1.

42

1.49

1.

57

1.47

Leuc

ine

2.62

2.

57

2.47

2.

56

2.53

2.

49

2.52

2.

70

2.48

62

Lysi

ne

2.05

1.

99

1.93

1.

92

1.89

1.

89

1.92

2.

07

1.89

Met

hion

ine

0.58

0.

57

0.50

0.

57

0.51

0.

55

0.50

0.

61

0.51

Orn

ithin

e 0.

03

0.02

0.

02

0.02

0.

02

0.02

0.

02

0.02

0.

02

Phen

ylal

anin

e 1.

64

1.59

1.

59

1.59

1.

62

1.54

1.

61

1.68

1.

60

Prol

ine

1.96

1.

74

1.67

1.

92

1.70

1.

81

1.68

1.

83

1.78

Serin

e 1.

53

1.49

1.

49

1.44

1.

45

1.38

1.

47

1.49

1.

41

Taur

ine

0.20

0.

22

0.18

0.

21

0.16

0.

20

0.16

0.

20

0.16

Thre

onin

e 1.

27

1.22

1.

18

1.20

1.

20

1.17

1.

19

1.27

1.

17

Tryp

toph

an

0.44

0.

46

0.40

0.

43

0.44

0.

41

0.47

0.

48

0.46

Tyro

sine

1.

01

0.98

1.

07

0.96

1.

10

1.06

1.

01

1.12

1.

08

Val

ine

1.59

1.

55

1.52

1.

55

1.53

1.

49

1.52

1.

65

1.50

1 D

iets

wer

e an

alyz

ed a

t Uni

vers

ity o

f Mis

sour

i-Col

umbi

a, A

gric

ultu

re E

xper

imen

t Sta

tion

Che

mic

al L

abor

ator

y.

63

Table 4 Response of juvenile Litopenaeus vannamei (Initial weight 0.15±0.01g, 46 days in trial 1; initial weight 0.20±0.01g, 35 days in trial 2) offered diets with different soybean ingredients at varying levels and w/o fermented yeast.

Trial Diet Final Biomass (g)

Final Mean Weight (g) WG3 (%) FCR2 Survival

(%)

Trial 1 n = 6

1 Basal 65.74a 4.83ab 3332ab 1.46bc 91.1a

2 LN1 53.51b 4.98ab 3463a 1.42bc 72.2bc

3 HN1 49.79b 5.08a 3359ab 1.38c 65.6c

4 LN2 49.98b 4.33bc 2831ab 1.65ab 77.8abc

5 HN2 49.37b 4.06c 2627b 1.75a 81.1abc

6 LN3 57.20ab 4.58abc 3128ab 1.54abc 83.3abc

7 HN3 58.60ab 4.34bc 3032ab 1.62abc 90.0ab

8 Basal+yeast 53.89b 4.75ab 3136ab 1.48bc 75.6abc

9 HN1+yeast 55.01ab 4.61abc 2926ab 1.53abc 80.0abc

P-value 0.0009 0.0001 0.0136 0.0002 0.0008

PSE1 1.0492 0.0592 66.1983 0.0215 1.6183

Trial 2 n = 6

1 Basal 36.25a 4.05ab 2005a 1.39cd 90.0

2 LN1 36.72a 4.02abc 2050a 1.41cd 91.7

3 HN1 31.78abc 3.90abc 1880ab 1.45bcd 81.7

4 LN2 27.72bc 3.60bcd 1731abc 1.59abc 78.3

5 HN2 27.32c 3.29d 1519c 1.74a 83.3

6 LN3 35.03ab 3.76abcd 1793abc 1.50bcd 93.3

7 HN3 31.98abc 3.49cd 1652bc 1.63ab 91.7

8 Basal+yeast 34.95ab 4.24a 1981ab 1.34d 83.3

9 HN1+yeast 33.68abc 3.68bcd 1733abc 1.54abcd 91.7

P-value 0.0003 <0.0001 <0.0001 <0.0001 0.2750

PSE1 0.6483 0.0479 31.2517 0.0192 1.9902 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight-initial weight)/initial weight*100%.

64

Values within a column with different letters are significantly different based on Tukey’s multiple range test.

65

Table 5 Principle component analysis of trypsin inhibitors and essential amino acids of diets.

PC1 PC2 PC3

TrypsinInhibitor -0.153 -0.013 0.836

Histidine 0.342 -0.095 -0.185

Isoleucine 0.346 0.105 0.145

Leucine 0.341 -0.152 0.219

Lysine 0.334 -0.205 -0.140

Cysteine 0.234 0.572 -0.225

Methionine 0.270 -0.495 0.095

Phenylalanine 0.316 0.333 0.093

Tryptophan 0.215 0.444 0.318

Threonine 0.342 -0.163 0.038

Valine 0.347 -0.089 0.084

Eigenvalue 7.821 1.302 1.093

% total variance 71.100 11.830 9.930

Cumulative eigenvalue 7.821 9.123 10.216

Cumulative % 71.10 82.93 92.86

66

Tab

le 6

Mul

tiple

line

ar r

egre

ssio

n of

wei

ght g

ain

(WG

) an

d fe

ed c

onve

rsio

n ra

tio (

FCR

) w

ith f

irst t

hree

prin

cipl

e co

mpo

nent

s (P

C1

PC2

and

PC3)

.

Tria

l M

odel

P-v

alue

and

R2

Para

met

er e

stim

ate

P-va

lue

for e

ach

varia

ble

PC1

PC2

PC3

PC1

PC2

PC3

1 W

G

P=0.

0095

R2 =0

.203

23

.85

-96.

10

-148

.75

P=0.

2433

P=

0.05

79

P=0.

0082

FCR

P<

0.00

01 R

2 =0.3

68

-0.0

11

0.02

9 0.

085

P=0.

1161

P=

0.07

98

P<0.

0001

2 W

G

P<0.

0001

R2 =0

.418

35

.39

-69.

78

-86.

83

P=0.

0004

P=

0.00

35

P=0.

0010

FCR

P<

0.00

01 R

2 =0.4

63

-0.0

26

0.04

5 0.

063

P<0.

0001

P=

0.00

33

P=0.

0003

67

Table 7 Pearson correlation coefficients of growth performances (final biomass, final mean weight, and weight gain), feed conversion ratio (FCR), and survival with trypsin inhibitors, methionine and protein levels of the diets. The first line of each cell is the value of correlation coefficient and the second line of each cell is P-value.

Trial Final

biomass

Final mean

weight

Weight

gain FCR Survival

Trial 1

Trypsin

inhibitors

-0.1350 -0.6063 -0.4081 0.5971 0.2647

0.3304 <0.0001 0.0022 <0.0001 0.0530

Methionine 0.1603 0.1850 0.1767 -0.1828 0.0064

0.2468 0.1805 0.2013 0.1858 0.9633

Lysine 0.2743 0.3561 0.3206 -0.3529 0.0080

0.0448 0.0082 0.0181 0.0089 0.9542

Trial 2

Trypsin

inhibitors

-0.4540 -0.5655 -0.5478 0.5964 -0.0661

0.0006 <0.0001 <0.0001 <0.0001 0.6351

Methionine 0.2926 0.5183 0.4694 -0.4950 -0.0493

0.0318 <0.0001 0.0003 0.0001 0.7232

Lysine 0.3851 0.6114 0.5536 -0.5915 -0.0233

0.0040 <0.0001 <0.0001 <0.001 0.8273

68

Tab

le 8

Tw

o-w

ay a

naly

sis

outp

ut o

f stre

ss a

nd im

mun

e re

spon

se o

f Pac

ific

whi

te s

hrim

p L.

van

nam

ei fe

d w

ith d

iffer

ent s

oy s

ourc

es

and

ferm

ente

d ye

ast p

rodu

ct le

vels

.

Soy

sour

ces

DV

A

(%)

THC

1 H

CC

2 G

CC

3 SG

CC

4 H

G5

HPC

V6

HP7

HPC

8 H

RB

A9

AU

soy

0 5.

891

4.04

0 0.

560

1.57

3 30

.643

12

.667

12

.229

44

.270

0.

0252

Schi

llnge

r1

0 5.

779

3.51

2 0.

504

1.33

6 30

.286

6.

900

12.4

78

44.8

60

0.02

47

AU

soy

0.13

6.

117

3.67

0 0.

200

1.45

0 30

.067

11

.545

12

.317

53

.462

0.

0230

Schi

llnge

r1

0.13

5.

767

2.29

0 0.

300

1.16

0 37

.938

5.

000

13.1

17

47.8

38

0.01

55

PSE

0.

1344

0.

224

1 0.

0277

0.

1437

0.

4852

0.

6090

0.

3271

1.

1782

0.

0003

Soy

sour

ces

Ferm

ente

d ye

ast

Soy

sour

ces *

Ferm

ente

d

yeas

t

0.65

10

0.08

1

4 0.

3114

0.

4141

0.

0384

0.

0028

0.

5982

0.

4489

0.

5534

0.83

64

0.08

4

2 0.

0004

0.

6069

0.

0631

0.

4374

0.

6702

0.

1220

0.

3988

0.82

21

0.36

8

4 0.

1915

0.

9297

0.

0346

0.

8407

0.

7567

0.

4249

0.

6072

Mod

el

0.95

95

0.09

4

1 0.

0022

0.

8009

0.

0089

0.

0233

0.

9023

0.

3067

0.

7134

1 TH

C: T

otal

hem

ocyt

e co

unt (

x106 /m

l).

2 HC

C: H

yalin

e ce

lls c

ount

(x10

6 /ml).

3 G

CC

: Gra

nula

r cel

ls c

ount

(x10

6 /ml).

4 S

GC

C: S

emi-g

ranu

lar c

ells

cou

nt (x

106 /m

l).

5 HG

: Hem

olym

ph g

luco

se (m

g/dl

). 6 H

PCV

: Hem

olym

ph p

acke

d ce

lls v

olum

e (%

). 7 H

P: H

emol

ymph

pro

tein

(mg/

ml).

69

8 HPC

: Hem

ocyt

e ph

agoc

ytic

cap

acity

(%).

9 HR

BA

: Hem

ocyt

e re

spira

tory

bur

st a

ctiv

ity (O

D).

70

CHAPTER IV

EVALUATION OF DRIED FERMENTED BIOMASS AS A FEED INGREDIENT IN

PLANT-BASED PRACTICAL DIETS FOR JUVENILE PACIFIC WHITE SHRIMP

Litopenaeus vannamei

1. Introduction

As shrimp consumption is expected to continue to increase globally, it is important to

develop sustainable alternative ingredients in shrimp diets to support the rapid expansion of the

shrimp industry (Achupallas et al., 2016). A range of traditional plant protein products from

agricultural production such as soybean meal (SBM) and corn gluten meal (CGM) have been

identified as appropriate alternative ingredients to complement or replace fish meal (FM) in

shrimp feeds. To further enhance the protein composition of SBM and CGM, various processes

have been utilized to create concentrates which have been used with good success in practical

shrimp feed formulations (Alam et al. 2005; Bauer et al. 2012; Fang et al. 2016; Paripatananont

et al. 2001; Qiu & Davis 2016a; b; c; d; Sookying & Davis 2012; Zhou et al. 2015). The

successful replacement of FM by soy- and corn-based products can result in reduced cost of feed

to a certain extent, however, the higher protein products are also somewhat expensive.

Coproducts of manufacturing processes have been gaining interests as alternative protein

sources in shrimp feeds as they have high nutritional value for less cost, because these are

derived from secondary streams of various commodity processing applications. Corn and wheat

coproducts such as distiller’s dried grains with solubles, fermented bacterial biomass, and

ethanol yeast are the most popular coproducts from the biofuels and ethanol industries.

Fermented bacterial biomass received considerable attention because of its well-known

71

properties of rapid growth and protein accretion in protein production (Kuhad et al. 1997;

Stringer 1982). Various strains of bacteria with a carbohydrate sugar source such as molasses,

sucrose, or glucose were utilized in the fermentation process for L-threonine (Melanie et al.

2015). This process results in a dried fermented biomass which is derived from the manufacture

of L-thereonine by fermentation using Escherichia coli. It has a high protein content (up to 800 g

kg-1) and good amino acid (AA) composition, which may serve as a potential alternative protein

ingredient in the aqua feed industry.

This DFB product has been confirmed to successfully replace FM and SBM in nursery

diets for pigs (Perez et al. 2011) and served as a substitution for FM in practical diets for Florida

pompano, Trachinotus carolinus (Melanie et al. 2015). However, information about the

application of DFB in practical shrimp feed is still limited. Therefore, the purposes of this study

were to determine the biological responses of Pacific white shrimp, L. vannamei to dietary DFB

supplementation as a replacement for FM or soy protein concentrate (SPC) w/o corn protein

concentrate (CPC). Additionally, the effects of two drying processes on nutritional value of the

meals to shrimp were assessed.

2. Material and Methods

2.1. Experimental design and diets

All test diets were formulated to be iso-nitrogenous and iso-lipidic (350 g kg-1 protein and

80 g kg-1 lipid). In trial 1, the basal diet was primarily composed of FM, SBM, corn gluten meal,

and whole wheat. Four experimental diets (DF0, DFB25, DFB50, and DFB100) were formulated

to utilize increasing levels (0, 25, 50, and 100 g kg-1) of DFB as a replacement of FM (Table 1).

In trial 2, the basal diet was primarily consisted of FM, SBM, corn protein concentrate (CPC),

72

and soy protein concentrate (SPC). Nine experiment diets were formulated to be supplemented

with increasing levels (0, 20, 40, 60, and 120 g kg-1) of SDFB and GDFB) to replace SPC w/o

CPC (Table 2).

Primary ingredients were analyzed at University of Missouri Agricultural Experiment

Station Chemical Laboratories (Columbia, MO, USA) for proximate and amino acid composition

(Table 3). All experimental diets were produced at the Aquatic Animal Nutrition Laboratory at

the School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University (Auburn, AL,

USA), using standard procedures for shrimp feeds. Briefly, diets were prepared by mixing the

pre-ground dry ingredients in a food mixer (Hobart, Troy, OH, USA) for 10–15 minutes. Hot

water was then blended into the mixture to obtain a consistency appropriate for pelleting. Diets

were pressure-pelleted using a meat grinder with a 3-mm die in trial and a 2.5-mm die in trial 2.

The moist pellets were then placed into a fan-ventilated oven (< 50 °C) overnight in order to

attain a moisture content of less than 10%. Dry pellets were crumbled, packed in sealed bags,

and stored in a freezer until use. The diets from trial 2 were analyzed at University of Missouri

Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA) for proximate

and amino acid composition (Table 4)

2.2. Growth trials

Two trials were conducted at the E.W. Shell Fisheries Research Station, Auburn

University (Auburn, AL, USA). Pacific white shrimp post larvae (PL) were obtained from

Shrimp Improvement Systems (Islamorada, Florida) and nursed in an indoor recirculating

system. PLs were fed a commercial feed (Zeigler Bros., Inc., Gardners, Pennsylvania, USA)

using an automatic feeder for ~1 week, and then switched to crumbled commercial shrimp feed

(Zeigler Bros., Inc., Gardners, Pennsylvania, USA) for ~1- 2 weeks.

73

In trial 1, the recirculating system consisted of 16 square tanks (340 L) connected to a

common reservoir, biological filter, bead filter, fluidized biological filter and recirculation pump.

Four replicate groups of shrimp (0.59 g initial mean weight; 10 shrimp / tank) were offered diets

using our standard feeding protocol over 6 weeks. Based on historic results, feed inputs were

pre-programmed assuming the shrimp would double their weight weekly up to one gram then

gain 0.8 g weekly with a feed conversion ratio (FCR) of 1.8. Daily allowances of feed were

adjusted based on observed feed consumption, weekly counts of the shrimp and mortality.

Consequently, for each tank in trial 1, a fixed ration of 1.5 g day-1 for the first and second week

and 2.1 g day-1 for the remaining culturing period was offered, partitioned in 4 feedings each

day.

In trial 2, the recirculating system consisted of 45 aquaria (80 L) connected to a common

reservoir, biological filter, bead filter, fluidized biological filter and recirculation pump. Four

replicate groups of shrimp (2.34 g initial mean weight, 10 shrimp / tank) were offered diets using

a standard feeding protocol over 6 weeks. Based on historic results, feed inputs were pre-

programmed assuming the shrimp would double their weight weekly up to one gram then gain

0.8-1.4 g weekly with a feed conversion ratio (FCR) of 1.8. Daily allowances of feed were

adjusted based on observed feed consumption, weekly counts of the shrimp and mortality.

Consequently, for each tank in trial 2, a fixed ration of 2.1 g day-1 for the first week, 2.6 g day-1

for the second week, 2.8 g day-1 for the third week, 3.3 g day-1 for the fourth and fifth week, and

3.6 g day-1 for the last week was allotted in 4 portions per day.

At the conclusion of each growth trial, shrimp were counted and group-weighed. Mean

final weight, FCR, WG, biomass, and survival were determined (Table 5 and Table 6). After

obtaining the final total weight of shrimps in each aquarium, four shrimps from each tank in trial

74

2 were frozen at -20 °C for subsequent determination of whole body composition. Proximate

composition of whole shrimp was analyzed by Midwest Laboratories (Omaha, NE, USA).

Protein retention was calculated as follows:

Protein retention efficiency (PRE, %) = (final weight × final protein content) - (initial weight ×

initial protein content) × 100 / protein offered.

2.3. Water quality monitoring

For both trials, dissolved oxygen (DO), water temperature and salinity were measured

twice daily using a YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA).

Hydrogen potential (pH) was measured twice weekly using a waterproof pHTestr30 (Oakton

instrument, Vernon Hills, IL, USA). Total ammonia-nitrogen (TAN) and nitrite were evaluated

every week using the methods described by Solorzano (1969) and Spotte (1979).

2.4. Statistical analysis

All the data were analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). Data from

trial 1 and 2 were analyzed using one-way ANOVA to determine significant differences (P <

0.05) among treatments followed by Tukey’s multiple comparison test to determine difference

between treatment means in each trial. The pooled standard errors were used across growth trials,

as the variance of each treatment is the same. Analysis of covariance (ANCOVA) was performed

to determine the importance of the processing methods (covariate) in trial 2.

75

3. Results

3.1. Water quality

In trial 1, DO, temperature, and salinity were maintained at 6.18 ± 0.41 mg L-1, 29.7 ± 0.8

°C, and 2.8 ± 0.4 ppt, respectively. In trial 2, DO, temperature, salinity, pH, TAN, and nitrite

were maintained at 6.94 ± 0.47 mg L-1, 28.5 ± 0.7 °C, 8.6 ± 0.7 ppt, 7.2 ± 0.4, 0.05 ± 0.05 mg L-

1, and 0.05 ± 0.05 mg L-1, respectively. Water quality conditions in both of the trials were

suitable for normal growth and survival of this species.

3.2. Growth trials

Performances of Pacific white shrimp offered diets with various DFB levels in Trials 1

and 2 are presented in Tables 5 and 6, respectively. In trial 1, final mean weight, WG and FCR

were not negatively affected when shrimp fed with diets contained up to 50 g kg-1 DFB as a

replacement of FM. However, a level of 100 g kg-1 DFB significantly decreased WG but

increased FCR. No significant differences were detected in final biomass (57% to 62.7%) or

survival (100%) across all the treatments.

In trial 2, shrimp fed with diet contained 20 g kg-1 GDFB performed the best across all

the treatments in terms of final mean weight, WG, and FCR. GDFB can be utilized up to 120 g

kg-1 to replace SPC and CPC without causing negative effects on growth performance and FCR.

However, dietary SDFB supplementation at 60 and 120 g kg-1 significantly reduced final mean

weight and WG of shrimp, while increased FCR compared to the treatment contained 20 g kg-1

GDFB. No significant impacts were detected in final biomass (80.4 to 92.2 g), survival (84 to 90

%), and PRE (27.5 to 35.9 %).

76

3.3. Whole body composition

Whole body proximate composition of shrimp when animals were offered diets contained

various DFB levels from two sources are presented in Table 7. Shrimp fed with diets

supplemented with 120 g kg-1 GDFB exhibited significantly lower body lipid level than those fed

with diets containing 40 and 120 g kg-1 SDFB. Significant differences were detected in carcass

moisture content among dietary treatments, however, Tukey’s multiple comparison did not

determine significant differences between the treatments, which may due to the variance. No

significant differences were observed in protein (676.2 to 756.4 g kg-1) or ash (83.1 to 119.4 g

kg-1) contents of whole shrimp body.

3.4. ANCOVA output

ANCOVA output of performance and proximate composition of whole shrimp body is

presented in Table 8. The main effect (DFB inclusion levels) and the covariate effect (processing

methods) had significant effects on final mean weight, WG, and FCR. A combined effect of DFB

inclusion levels and processing methods was observed on the lipid content of whole shrimp

body. No differences were detected in survival, PRE, and moisture, protein, and ash content of

whole shrimp body.

4. Discussion

Feed represents over 50% of the cost for aquaculture producers, which is an important

economic part that can be improved upon. Identification of suitable protein sources as FM

substitutes in shrimp feeds may result in reduced feed costs and foster an economically feasible,

expanded, and sustainable shrimp industry. The analyzed proximate composition indicates that

77

DFB has a higher protein content (790 and 805.1 g kg-1) than FM (627.8 g kg-1), SPC (649.3 g

kg-1), and CPC (780.7 g kg-1). When FM is replaced with alternative protein sources, it is critical

to balance the amino acid profile in the diets. Methionine and lysine are two essential amino

acids (AA) that are typically most limiting AA in the plant-derived byproducts. Concentrations

of these and other AA in DFB are similar to FM and much higher than in SPC and CPC (Table

3). As a consequence, AA levels in diets supplemented with DFB were higher than those of the

basal diet (Table 4).

In trial 1, data indicates that DFB can be utilized up to 50 g kg-1 in shrimp diets to

replace FM without compromising the growth. However, a significant reduction in WG and

increase in FCR were observed when shrimp were fed with the diet supplemented with 100 g kg-

1 DFB. A number of studies have evaluated the feasibility of inclusion of bacterial biomass as a

substitute for FM. Only a few of them were conducted on coproducts from AA production such

as fermented bacterial biomass. The same product (DFB) has been demonstrated to be suitable

for complete replacement of FM (120 g kg-1) in Florida pompano Trachinotus carolinus diets

containing 8% poultry byproduct meal (Rhodes et al. 2015) and nursery pig diets (Perez et al.

2011), without resulting in negative effects on growth of experiment animals or FCR. A similar

dried bacterial biomass from L-lysine manufacture using fermenting bacteria, Micrococcus

glutamicus was used up to 100 g kg-1 as a replacement of FM without causing negative effects on

the growth of tilapia, Oreochromis mossambicus; however, significant reduction on growth and

increase in FCR were detected when lysine biomass was supplemented at higher levels (150 and

200 g kg-1) in tilapia diets (Davies & Wareham 1988).

Aside from AA bacterial byproducts, using methane-oxidizing bacteria as protein and AA

sources in aquatic animal nutrition recently received considerable attention due to the abundant

78

supply, cheap transportation, and reasonable cost of natural gas (Øverland et al. 2010). A

bacterial protein meal (BPM) consisting of M. capsulatus, Alcaligenes acidovorans, Bacillus

brevis and B. firnus was produced in a process that converts natural gas into protein (Aas et al.

2007). Aas et al. (2006a) reported that BPM can be utilized up to 360 g kg-1 to replace FM in the

diets for Atlantic salmon, Salmo salar, without compromising growth. Similar results were

demonstrated by Berge et al. (2005) and Storebakken et al. (2004) who documented that BPM

can be supplemented in S. salar diets at 200 and 193 g kg-1, respectively. The inclusions of BPM

up to 270 g kg-1 were also confirmed to be successfully applied in the diets for rainbow trout

Oncorhynchus mykiss as a substitute for FM (Aas et al. 2006b). In contrast, Aas et al. (2007)

indicated that BPM can be used up to 90 g kg-1 in the diets for Atlantic halibut Hippoglossus

hippoglossus but supplementation of BPM at 180 g kg-1 caused growth depression in this

species.

Results from the researches listed above demonstrated that partial replacement of FM by

bacterial biomass proteins are appropriate for several aquatic species. However, the effects of

high inclusion levels of bacterial biomass proteins in diets for aquatic animals are still

contradictory among those studies. Variations among these researches may be attributed to

various strains utilized to produce the bacterial biomass proteins, processing of the meal, and

different species of experimental animals. The failure of complete FM replacement by DFB in

the present study may due to the palatability or nutritional imbalances in the diet devoid of FM.

Soy protein concentrate is produced through a series of different extraction and

precipitation process from high-quality de-hulled soybeans, and its crude protein content can

reach up to 700 g kg-1 (Sookying & Davis 2012). Corn protein concentrate (CPC) is produced

through wet milling and refined to contain up to 800 g kg-1 crude protein (AAFCO 2007). The

79

application of SPC in shrimp feeds were demonstrated to be feasible in multiple studies (Alam et

al. 2005; Bauer et al. 2012; Paripatananont et al. 2001; Sookying & Davis 2012). Inclusion

levels of CPC at 47-60 g kg-1 were widely applied in practical diets for Pacific white shrimp as a

good source for protein and methionine (Fang et al. 2016; Zhou et al. 2015; Qiu & Davis 2016a;

b; c; d). Based on the information provided by trial 1 in the present study, a follow up growth

trial (trial 2) further explored different inclusion levels (0, 20, 40, 60, 120 g kg-1) of DFB

products produced by two different processing methods (SDFB and GDFB) as a substitution for

SPC w/o CPC in a low FM-based diet. Results indicated that GDFB can be utilized up to 120 g

kg-1 to replace SPC and CPC without causing negative effects on growth performance and FCR.

However, significantly reduced final mean weight and WG as well as increased FCR were

detected when dietary SDFB were supplemented at 60 and 120 g kg-1. Analysis of covariance

detected a significant effect of the processing method on the final biomass, final mean weight,

WG of shrimp and FCR. Shrimp fed with GDFB performed significantly better in terms of

growth than those fed with SDFB. The research on bacterial biomass proteins for utilization as

replacements for SPC and CPC in aquatic animal feeds is still limited. Based on information

provided in the FM replacement researches conducted in the current study and by many other

researchers, the successfully replacement of SPC w/o CPC by GDFB could be easily understood

because of the balanced amino acid profile and suitable palatability. However, the significant

reduction on the shrimp performances resulted from 60 and 120 g kg-1 SDFB supplementation

indicated that spray drying method may not be the most appropriate processing option for

proteinaceous bacterial meal.

In the present study, PRE was not significantly affected when either SDFB or GDFB

were utilized up to 120 g kg-1. Similarly, Aas et al. (2006) documented that inclusion of BPM up

80

to 270 g kg-1 did not affect PRE in rainbow trout. Also in agreement, Aas et al. (2007) and Berge

et al. (2005) reported that PRE in Atlantic salmon was not significantly affected when BPM was

supplemented up to 180 and 200 g kg-1 as a replacement of FM. PRE was determined by a

number of factors including dietary protein level, feed intake, final weight and initial weight of

animals as well as the final and initial protein content of animals (Halver & Hardy, 2002). In the

current study, no significant differences were found in dietary protein levels, feed offered to the

shrimp, initial weight of shrimp, and protein content of shrimp carcass. The significantly reduced

final weight of shrimp fed with the diet supplemented with 60 g kg-1 SDFB resulted in

comparatively lower PRE observed in this treatment.

With regards to whole body proximate composition, lipid content in shrimp fed the diet

containing 120 g kg-1 GDFB was significant lower than those fed with diets supplemented with

40 and 120 g kg-1 SDFB. No significant differences were detected in carcass protein or ash

content. Likewise, Aas et al. (2006b) reported that no significant differences were detected in the

contents of crude lipid, crude protein, or ash in carcass of rainbow trout when BPM were utilized

up to 270 g kg-1 in the diet. In addition, other published work also documented that dietary

bacteria protein meal had no significant effect on proximate composition (protein, lipid, or ash)

of Atlantic halibut (Aas et al. 2007) and Atlantic salmon (Aas et al. 2006a; Berge et al. 2005;

Storebakken et al. 2004). In the present experiment, analysis of covariance indicated that the

combined effect of the DFB source and level significantly affected the whole lipid content of

shrimp. Generally, shrimp fed with diets containing SDFB had a relatively higher lipid level than

those fed with GDFB. The carcass lipid content decreased as the inclusion level of GDFB

increased, which may be attributed to relatively lower energy availability in this ingredient.

81

5. Conclusion

Under the reported conditions of the study, the use of DFB can partially replace FM up to

50 g kg-1 without causing negative effects on the growth performance of Pacific white shrimp.

However, completely replacement of FM (100 g kg-1) by DFB resulted in growth depression

which may due to palatability or nutrient imbalances. The use of GDFB as a substitution for SPC

w/o CPC did not significantly affect growth of shrimp. However, the inclusion of SDFB at 60

and 120 g kg-1 decreased growth of shrimp indicating a negative effect of spray drying. The

results in the current study demonstrate that GDFB is a good protein source which can be

incorporated in practical shrimp feed formulations.

82

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Association of American Feed Control Officials, INC., Rockville, Maryland.

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nutrient utilisation in Atlantic salmon (Salmo salar) fed diets containing a bacterial

protein meal. Aquaculture 259, 365-376.

Aas, T.S., Hatlen, B., Grisdale-Helland, B., Terjesen, B.F., Bakke-McKellep, A.M. & Helland,

S.J. (2006b) Effects of diets containing a bacterial protein meal on growth and feed

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Aas, T.S., Hatlen, B., Grisdale‐Helland, B., Terjesen, B.F., Penn, M., Bakke‐McKellep, A.M. &

Helland, S.J. (2007) Feed intake, growth and nutrient utilization in Atlantic halibut

(Hippoglossus hippoglossus) fed diets containing a bacterial protein meal. Aquac. Res. 38,

351-360.

Achupallas, J.M., Zhou, Y., & Davis D.A (2016). Use of grain distillers dried yeast in practical

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Alam, M.S., Teshima, S.I., Koshio, S., Ishikawa, M., Uyan, O., Hernandez, L.H.H. & Michael,

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19.

Bauer, W., Prentice-Hernandez, C., Tesser, M.B., Wasielesky, W., & Poersch, L.H. (2012)

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the pacific white shrimp Litopenaeus vannamei. Aquaculture 342, 112-116.

83

Berge, G.M., Baeverfjord, G., Skrede, A. & Storebakken, T. (2005) Bacterial protein grown on

natural gas as protein source in diets for Atlantic salmon (Salmo salar) in saltwater.

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genetically modified soybean varieties as ingredients in practical diets for Litopenaeus

vannamei. Aquaculture 451, 178-185.

Kuhad, R.C., Singh, A., Tripathi, K.K., Saxena, R.K. & Eriksson, K.E.L. (1997) Microorganisms

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bacteria products as feed ingredients for monogastric animals. Arch. Anim. Nutr. 64, 171-

189.

Paripatananont, T., Boonyaratpalin, M., Pengseng, P. & Chotipuntu, P. (2001) Substitution of

soy protein concentrate for fishmeal in diets of tiger shrimp Penaeus monodon. Aquac.

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can replace protein from fish and soybean meals in nursery diets. J. Anim. Sci. 89, E-

Suppl. 1/J: 328.

Qiu, X. & Davis, D.A. (2016a) Effects of dietary phytase supplementation on growth

performance and apparent digestibility coefficients of Pacific white shrimp Litopenaeus

vannamei. Aquac. Nutr. doi: 10.1111/anu.12462.

84

Qiu, X. & Davis, D.A. (2016b) Effects of dietary carbohydrase supplementation on performance

and apparent digestibility coefficients in Pacific white shrimp Litopenaeus vannamei. J.

World Aquac. Soc. doi: 10.1111/jwas.12361.

Qiu, X. & Davis, D.A. (2016c) Evaluation of flash dried yeast as a nutritional supplement in

plant-based practical diets for Pacific white shrimp, Litopenaeus vannamei. Aquac. Nutr.

doi: 10.1111/anu.12499.

Qiu, X., Buentello, A., Shannon, R., Mustafa, A., Abebe, A. & Davis, D.A. (2016d) Evaluation

of three non-genetically modified soybean cultivars as ingredients and a yeast-based

additive as a supplement in practical diets for Pacific white shrimp, Litopenaeus

vannamei. Aquac. Nutr. In press.

Rhodes, M.A., Zhou, Y. & Davis, D.A. (2015) Use of dried fermented biomass as a fish meal

replacement in practical diets of Florida pompano, Trachinotus carolinus. J. Appl. Aquac.

27, 29-39.

Sookying, D. & Davis, D.A. (2012) Use of soy protein concentrate in practical diets for Pacific

white shrimp (Litopenaeus vannamei) reared under field conditions. Aquac. Int. 20, 357-

371.

Solorzano, L. (1969) Determination of ammonia in natural waters by the phenolhypochlorite

method. Limnol. Oceanogr. 14, 799-801.

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edition. Wiley, New York.

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85

Storebakken, T., Baeverfjord, G., Skrede, A., Olli, J.J. & Berge, G.M. (2004) Bacterial protein

grown on natural gas in diets for Atlantic salmon, Salmo salar, in freshwater.

Aquaculture 241, 413-425.

Zhou, Y.G., Davis, D.A. & Buentello, A. (2015) Use of new soybean varieties in practical diets

for the Pacific white shrimp, Litopenaeus vannamei. Aquac. Nutr. 21, 635-643.

86

Table 1 Composition (g kg-1 as is) of test diets utilized in trial 1.

Ingredient Diet code

DFB0 DFB25 DFB50 DFB100 Soybean meal1 350.0 350.0 350.0 350.0 Fish meal2

160.0 129.0 98.0 37.0 Corn gluten meal3 50.0 50.0 50.0 50.0 Whole wheat4

340.5 340.5 340.5 340.5 Dried fermented biomass5

0 25.0 50.0 100.0 Fish oil2 47.3 49.2 51.1 54.7 Trace mineral premix6 5.0 5.0 5.0 5.0 Vitamin premix7 18.0 18.0 18.0 18.0 Choline chloride4 2.0 2.0 2.0 2.0 Stay C8 1.0 1.0 1.0 1.0 Lecithin9 10.0 10.0 10.0 10.0 Cholesterol4 0.5 0.5 0.5 0.5 Corn starch4 5.7 6.3 7.4 8.3 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Omega Protein Inc., Huston TX, USA. 3 Grain Processing Corporation, Muscatine, IA, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5 Proplex T, Archer Daniels Midland Company, Chicago, IL, USA. 6 Trace mineral premix (g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 7 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 8 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 9 The Solae Company, St. Louis, MO, USA.

87

Tab

le 2

Com

posi

tion

(g k

g-1 a

s is)

of t

est d

iets

util

ized

in tr

ial 2

.

Ingr

edie

nt

Die

t cod

e

Bas

al

SDFB

20

SDFB

40

SDFB

60

SDFB

12

0 G

DFB

20

GD

FB40

G

DFB

60

GD

FB12

0 Fi

sh m

eal1

60.0

60

.0

60.0

60

.0

60.0

60

.0

60.0

60

.0

60.0

So

ybea

n m

eal2

382.

0 38

2.0

382.

0 38

2.0

382.

0 38

2.0

382.

0 38

2.0

382.

0 W

hole

whe

at3

280.

0 28

0.0

280.

0 28

0.0

280.

0 28

0.0

280.

0 28

0.0

280.

0 C

orn

prot

ein

conc

entra

te4

60.0

60

.0

60.0

60

.0

0.0

60.0

60

.0

60.0

0.

0 So

y pr

otei

n co

ncen

trate

5 74

.0

50.0

26

.0

0.0

0.0

50.0

26

.0

0.0

0.0

Drie

d fe

rmen

ted

biom

ass5

0.0

20.0

40

.0

60.0

12

0.0

20.0

40

.0

60.0

12

0.0

Fish

oil1

53.5

52

.6

51.6

50

.6

48.9

52

.6

51.6

50

.6

48.9

Tr

ace

min

eral

pre

mix

6 5.

0 5.

0 5.

0 5.

0 5.

0 5.

0 5.

0 5.

0 5.

0 V

itam

in p

rem

ix7

18.0

18

.0

18.0

18

.0

18.0

18

.0

18.0

18

.0

18.0

C

holin

e ch

lorid

e8 2.

0 2.

0 2.

0 2.

0 2.

0 2.

0 2.

0 2.

0 2.

0 St

ay C

9 1.

0 1.

0 1.

0 1.

0 1.

0 1.

0 1.

0 1.

0 1.

0 M

ono-

dica

lciu

m p

hosp

hate

10

25.0

25

.5

26.5

27

.5

28.0

25

.5

26.5

27

.5

28.0

Le

cith

in11

10

.0

10.0

10

.0

10.0

10

.0

10.0

10

.0

10.0

10

.0

Cho

lest

erol

8 0.

8 0.

8 0.

8 0.

8 0.

8 0.

8 0.

8 0.

8 0.

8 C

orn

star

ch8

28.7

33

.1

37.1

43

.1

44.3

33

.1

37.1

43

.1

44.3

1 O

meg

a Pr

otei

n In

c., H

usto

n, T

X, U

SA.

2 D

e-hu

lled

solv

ent e

xtra

ct so

ybea

n m

eal,

Bun

ge L

imite

d, D

ecat

ur, A

L, U

SA.

3 Bob

s Red

Mill

, Milw

auki

e, O

R, U

SA.

4 Em

pyre

al®

75,

Car

gill

Cor

n M

illin

g, C

argi

ll, In

c., B

lair,

NE,

USA

. 5 Pr

ople

x T,

Arc

her D

anie

ls M

idla

nd C

ompa

ny, C

hica

go, I

L, U

SA.

88

6 Tr

ace

min

eral

pre

mix

(g/

100g

pre

mix

): C

obal

t ch

lorid

e, 0

.004

; C

upric

sul

fate

pen

tahy

drat

e, 0

.550

; Fe

rrou

s su

lfate

, 2.

000;

M

agne

sium

sulfa

te a

nhyd

rous

, 13.

862;

Man

gane

se su

lfate

mon

ohyd

rate

, 0.6

50; P

otas

sium

iodi

de, 0

.067

; Sod

ium

sele

nite

, 0.0

10; Z

inc

sulfa

te h

epta

hydr

ate,

13.

193;

Alp

ha-c

ellu

lose

, 69.

664.

7 V

itam

in p

rem

ix (g

/kg

prem

ix):

Thia

min

.HC

L, 4

.95;

Rib

ofla

vin,

3.8

3; P

yrid

oxin

e.H

CL,

4.0

0; C

a-Pa

ntot

hena

te, 1

0.00

; Nic

otin

ic a

cid,

10

.00;

Bio

tin, 0

.50;

fol

ic a

cid,

4.0

0; C

yano

coba

lam

in, 0

.05;

Ino

sito

l, 25

.00;

Vita

min

A a

ceta

te (

500,

000

IU/g

), 0.

32;

Vita

min

D3

(1,0

00,0

00 IU

/g),

80.0

0; M

enad

ione

, 0.5

0; A

lpha

-cel

lulo

se, 8

56.8

1.

8 M

P B

iom

edic

als I

nc.,

Solo

n, O

H, U

SA.

9 Sta

y C

®, (

L-as

corb

yl-2

-pol

ypho

spha

te 3

5% A

ctiv

e C

), D

SM N

utrit

iona

l Pro

duct

s., P

arsi

ppan

y, N

J, U

SA.

10 J.

T. B

aker

®, M

allin

ckro

dt B

aker

, Inc

., Ph

illip

sbur

g, N

J, U

SA.

11 Th

e So

lae

Com

pany

, St.

Loui

s, M

O, U

SA.

89

Table 3 Proximate composition, and amino acid profile of dried fermented biomass used in trial 1 (DFB) and 2 (SDFB and GDFB have identical nutrient compositions), fish meal (FM), soy protein concentrate (SPC), and corn protein concentrate (CPC).

Composition1 (g kg-1 as is) DFB S/GDFB FM SPC CPC

Crude protein 805.1 790.0 627.8 649.3 780.7

Moisture 47.8 34.5 79.9 85.5 81.6

Crude fat 10.0 49.0 105.6 0 2.0

Crude fiber - 8.0 0 3.5 0.9

Ash - 27.0 187.5 6.3 1.0

Alanine 60.6 55.3 39.1 26.3 64.2

Arginine 52.3 49.5 36.8 44.4 22.5

Aspartic acid 85.5 78.5 53.4 68.4 42.9

Cysteine 8.2 9.0 4.7 8.0 13.0

Glutamic acid 98.4 92.4 74.7 104.4 146.8

Glycine 39.1 35.9 48.8 25.5 19.5

Histidine 18.8 17.8 16.3 17.7 14.8

Isoleucine 42.4 39.6 24.2 29.5 29.6

Leucine 76.5 70.9 42.1 50.8 129.7

Lysine 53.3 49.8 46.7 38.8 11.4

Methionine 22.2 20.9 16.1 8.3 18.0

Phenylalanine 37.6 33.3 23.9 33.3 48.0

Proline 30.9 30.1 30.8 32.9 73.1

Serine 26.9 24.3 21.1 27.1 38.0

Threonine 41.4 36.7 24.1 23.8 24.8

Tryptophan 10.8 9.9 6.2 8.6 3.7

Tyrosine 30.3 28.4 16.7 25.0 42.4

Valine 53.3 50.8 29.9 29.4 32.3 1 Ingredients were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

90

Tab

le 4

Pro

xim

ate

com

posi

tion

and

amin

o ac

id p

rofil

e of

the

test

die

ts u

sed

in tr

ial 2

.

Com

posi

tion1

(g k

g-1 a

s is)

B

asal

SD

FB20

SD

FB 4

0 SD

FB 6

0 SD

FB 1

20

GD

FB20

G

DFB

40

GD

FB60

G

DFB

120

Cru

de p

rote

in

364.

9 36

9.6

365

365.

6 36

5.7

362.

1 36

5 36

5.4

369

Moi

stur

e 54

.9

50.8

63

.7

63.9

72

.7

51.3

51

.9

59.1

55

.6

Cru

de fa

t 10

2 86

.1

89.5

90

.6

86.1

88

.3

85.4

91

.4

90.2

C

rude

fibe

r 31

.7

33.1

31

.3

31.1

27

.5

35.2

33

.3

32.3

33

.1

Ash

67

.7

68.7

66

.8

67.2

68

.4

67.1

67

.2

63.9

68

.5

Ala

nine

18

.2

19.1

19

.2

19.5

19

.0

18.2

19

.2

19.6

19

.2

Arg

inin

e 22

.3

22.6

21

.8

21.4

23

.1

21.9

22

.0

21.8

23

.0

Asp

artic

Aci

d 33

.3

34.2

32

.9

32.3

34

.6

32.8

33

.1

32.9

34

.7

Cys

tein

e 5.

0 5.

0 4.

8 4.

7 4.

5 4.

6 4.

8 4.

7 4.

3 G

luta

mic

Aci

d 70

.8

72.0

69

.5

68.1

64

.9

69.0

69

.8

68.9

64

.9

Gly

cine

16

.8

17.1

17

.2

17.7

19

.3

16.7

17

.1

17.3

19

.0

His

tidin

e 8.

7 9.

0 8.

6 8.

4 8.

6 8.

5 8.

6 8.

5 8.

6 Is

oleu

cine

16

.2

16.8

16

.3

16.0

16

.7

16.0

16

.2

16.0

16

.3

Leuc

ine

31.0

32

.5

32.1

31

.6

28.8

30

.8

32.2

32

.1

28.8

Ly

sine

20

.7

21.0

20

.5

20.5

23

.0

20.6

20

.8

20.8

22

.8

Met

hion

ine

6.0

6.4

6.4

6.6

6.7

6.2

6.4

6.5

6.6

Phen

ylal

anin

e 18

.5

19.0

18

.4

18.0

17

.5

18.2

18

.7

18.3

17

.4

Prol

ine

23.0

22

.9

22.3

22

.3

20.0

22

.1

23.7

22

.0

20.0

Se

rine

15.3

15

.5

15.1

15

.1

15.0

15

.6

15.8

16

.1

15.8

Th

reon

ine

12.8

13

.5

13.3

13

.4

14.5

13

.3

13.7

13

.9

15.0

Tr

ypto

phan

4.

7 4.

5 4.

7 4.

8 5.

2 4.

8 4.

7 5.

0 5.

2 Ty

rosi

ne

12.7

13

.1

13

12.7

12

.4

12.6

13

.2

13.1

12

.3

91

Val

ine

18.0

18

.8

18.4

17

.7

18.6

17

.8

18.1

17

.6

19.3

1 D

iets

wer

e an

alyz

ed a

t Uni

vers

ity o

f Mis

sour

i Agr

icul

tura

l Exp

erim

ent S

tatio

n C

hem

ical

Lab

orat

orie

s (C

olum

bia,

MO

, USA

).

92

Table 5 Performance of juvenile shrimp L. vannamei (Initial weight 0.59 g) offered diets with

different dried fermented biomass (DFB) levels (0, 25, 50, and 100 g kg-1) for six weeks in trial

1.

Diet Final biomass (g) Final mean weight (g) WG3 (%) FCR2 Survival (%)

DFB0 62.7 6.3 970.0a 1.52b 100

DFB25 61.8 6.2 944.6ab 1.55ab 100

DFB50 58.5 5.9 884.9ab 1.64ab 100

DFB100 57.0 5.7 845.4b 1.70a 100

PSE1 0.8047 0.0737 14.0715 0.0212

P-value 0.0812 0.0493 0.0337 0.0395 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - initial weight) / initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

93

Tab

le 6

Per

form

ance

of

juve

nile

shr

imp

L. v

anna

mei

(In

itial

wei

ght 2

.34

g) w

hen

shrim

p w

ere

offe

red

diet

s w

ith d

iffer

ent l

evel

s (0

, 20

, 40,

60,

and

120

g k

g-1) o

f spr

ay d

ry (S

) and

gra

nula

r (G

) drie

d fe

rmen

ted

biom

ass

(DFB

) for

six

wee

ks in

tria

l 2.

Die

t Fi

nal b

iom

ass

(g)

Fina

l mea

n w

eigh

t (g)

W

G3 (%

) FC

R2

Surv

ival

(%)

PRE4

Bas

al

89.0

10

.4a

338.

9ab

1.61

b 86

.0

34.9

SDFB

20

82.6

9.

6ab

314.

2abc

1.78

ab

86.0

31

.2

SDFB

40

80.9

9.

7ab

319.

6abc

1.76

ab

84.0

30

.4

SDFB

60

80.4

8.

9b 28

1.7c

1.97

a 90

.0

27.5

SDFB

120

78.6

9.

4ab

302.

1bc

1.84

ab

84.0

30

.4

GD

FB20

89

.6

10.7

a 35

9.3a

1.53

b 84

.0

35.9

GD

FB40

86

.3

10.3

ab

338.

0abc

1.65

ab

84.0

33

.2

GD

FB60

92

.2

10.5

a 34

7.1ab

1.

57b

88.0

32

.5

GD

FB12

0 85

.0

9.9ab

31

6.9ab

c 1.

70ab

86

.0

31.1

PSE1

1.68

04

0.13

24

5.47

17

0.03

13

1.53

48

0.79

87

P-va

lue

0.17

77

0.00

21

0.00

22

0.00

19

0.92

57

0.07

04

1 PSE

: Poo

led

stan

dard

err

or.

2 FC

R: F

eed

conv

ersi

on ra

tio =

Fee

d of

fere

d / (

Fina

l wei

ght -

Initi

al w

eigh

t).

3 W

G: W

eigh

t gai

n =

(Fin

al w

eigh

t - in

itial

wei

ght)

/ ini

tial w

eigh

t × 1

00%

. 4 P

RE:

Pro

tein

rete

ntio

n ef

ficie

ncy

= (f

inal

wei

ght ×

fina

l pro

tein

con

tent

) - (i

nitia

l wei

ght ×

initi

al p

rote

in c

onte

nt) ×

100

/ pr

otei

n in

take

. V

alue

s w

ithin

a c

olum

n w

ith d

iffer

ent s

uper

scrip

ts a

re s

igni

fican

tly d

iffer

ent b

ased

on

Tuke

y’s

mul

tiple

rang

e te

st.

94

Table 7 Proximate analysis (g kg-1) of whole shrimp body when shrimp were offered diets with different levels (0, 20, 40, 60, and 120 g kg-1) of spray dry (S) and granular (G) dried fermented biomass (DFB) for six weeks in trial 2.

Diet Protein2 Moisture Lipid2 Ash2

Basal 737.0 757.2 83.1ab 106.0

SDFB20 726.2 750.8 84.2ab 111.0

SDFB40 744.4 768.8 93.6a 107.7

SDFB60 704.5 755.2 75.8ab 102.5

SDFB120 756.4 769.2 95.6a 83.1

GDFB20 715.4 749.4 88.0ab 102.1

GDFB40 723.5 754.2 81.5ab 94.6

GDFB60 676.2 750.8 72.2ab 118.3

GDFB120 718.4 756.4 67.4b 119.4

PSE1 10.4656 2.1546 2.4667 4.3119

P-value 0.4519 0.0390 0.0129 0.2364 1 PSE: Pool standard error. 2 Dry weight basis. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

95

Table 8 Analysis of covariance (ANCOVA) output of performance and proximate composition of whole shrimp body data in trial 2.

Parameters

Adjusted LSmeans P-value

SDFB GDFB Processing

method Level

Processing

method*Level Model

Performances

Final biomass 82.3 88.4 0.0081 0.0673 0.4306 0.0014

Final mean weight 9.6 10.3 0.0003 0.0106 0.5979 0.0003

WG1 311.3 340.0 0.0011 0.0103 0.7981 0.0010

FCR1 1.79 1.61 0.0003 0.0180 0.4874 0.0004

Survival 86.0 85.6 0.8459 0.9699 0.6781 0.9750

PRE1 31.8 32.6 0.5000 0.8541 0.5727 0.8445

Proximate composition

Moisture 760.2 753.7 0.0544 0.1421 0.2434 0.0706

Protein 733.7 714.1 0.2164 0.9737 0.4229 0.5325

Lipid 86.5 78.5 0.0353 0.3680 0.0089 0.0090

Ash 102.1 108.1 0.3588 0.6944 0.0219 0.0992 1 WG: weight gain. FCR: feed conversion ratio. PRE: protein retention efficiency.

96

CHAPTER V

EVALUATION OF A NOVEL BACTERIAL BIOMASS AS A SUBSTITUTION FOR

SOYBEAN MEAL IN PLANT-BASED PRACTICAL DIETS FOR PACIFIC WHITE

SHRIMP Litopenaeus vannamei

1. Introduction

Soybean meal (SBM) is usually considered as the most reliable ingredient and cost-

effective protein source in shrimp feed because of its worldwide availability, low price, relatively

balanced amino acid profile, and consistent composition (Amaya et al., 2007; Davis and Arnold,

2000). The dietary SBM inclusions in practical shrimp feeds have been well defined in a number

of studies (Amaya et al., 2007; Roy et al., 2009; Sookying and Davis, 2011). However, as the

popularity of SBM utilized as a primary protein ingredient in animal feed formulation increased,

the price of SBM has risen from $175 per metric ton in the year 2000 to over $500 per metric ton

in 2014 (Index Mundi, 2015). Therefore, it would be necessary for us to explore novel

ingredients that are more economical and sustainable as shrimp culture has become an expanded

and intensified economic activity.

The rapid growth and high protein content of bacteria in protein production has led to

considerable attention on the potential of the application of microbial protein in animal

production. One of the attractive substrates used to produce bacterial protein is methane, which is

the main component of natural gas and distributed widely in nature (Dalton, 2005; Hanson and

Hanson, 1996). The abundant supply, cheap transportation, and reasonable cost of natural gas,

indicated that protein production from natural gas could be realistic on a large scale (Øverland et

al., 2010). Moreover, owing to the low water and no fertile soil requirements, the cultivation of

97

bacterial protein does not compete with agriculture lands and even can be achieved in dry

climates.

A number of researches have been conducted to evaluate a bacterial protein produced

from mainly methane by natural gas fermentation as a protein ingredient for several fish species

including Atlantic salmon Salmo salar (Aas et al., 2006a; Berge et al., 2005; Storebakken et al.,

1998; 1999; 2004), rainbow trout Oncorhynchus mykiss (Aas et al., 2006b; Øverland et al.,

2006), and Atlantic halibut Hippoglossus hippoglossus (Aas et al., 2007). The feeding

experiments in most of these publications recommended that the bacterial protein derived from

natural gas can be utilized as a sustainable protein source in aquaculture production.

The bacterial biomass evaluated in the present study is derived from Methylobacterium

extorquens. The application of this product as a protein source in commercial type of shrimp feed

is still limited. Therefore, the purposes of this project are to determine the growth response of

Pacific white shrimp juveniles to increasing bacterial biomass inclusion levels as a substitution

for soybean meal and determine apparent digestibility values for bacterial biomass as compared

to other traditional protein sources.

2. Material and Methods

2.1. Experimental design and diets

Three trials were conducted to evaluate the biological response of shrimp to BB in soy-

based diets in terms of the growth. In the trial 1 and 2, test diets were formulated to be

isonitrogenous and isolipidic (35% protein and 8% lipid). In trial 1, three experimental diets

(T1D1 - T1D3) were formulated to contain increasing levels (0, 6, and 12%) of BB as a

replacement of SBM (Table 1). In trial 2, to confirm the results in trial 1 and investigate the

98

effects of low inclusion levels of BB, six experimental diets (T2D1 - T2D6) were formulated to

supplement with increasing levels (0, 1, 2, 4, 6, and 12%) of BB as a replacement of SBM (Table

2). In trial 3, five experimental diets (T3D1 - T3D5) were formulated (Table 3). T3D1, T3D2, and

T3D4 are the same as diets in trial 2 that utilized 0, 6, and 12% BB to replace SBM. Whereas

T3D3 and T3D5 utilized BB to replace the same ratio of SBM as T3D2 and T3D4, respectively, on

a digestible protein basis. Additionally, a reference diet (Table 4) was utilized to determine

digestibility coefficients in conjunction with 1% chromic oxide as an inert marker and 70:30

replacement strategy.

Primary ingredients were analyzed at University of Missouri Agricultural Experiment

Station Chemical Laboratories (Columbia, MO, USA) for proximate and amino acid composition

(Table 5). All experimental diets were produced at the Aquatic Animal Nutrition Laboratory at

the School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University (Auburn, AL,

USA) using the standard procedures for the shrimp feeds described by Qiu and Davis, (2016).

Briefly, diets were prepared by mixing the pre-ground dry ingredients in a food mixer (Hobart,

Troy, OH, USA) for 10–15 minutes. Hot water was then blended into the mixture to obtain a

consistency appropriate for pelleting. Diets were pressure-pelleted using a meat grinder with a

2.5-mm die. The wet pellets were then placed into a fan-ventilated oven (< 50 °C) overnight in

order to attain a moisture content of less than 10%. Dry pellets were crumbled, packed in sealed

bags, and stored in a freezer until use. The diets were analyzed at University of Missouri

Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA) for proximate

and amino acid composition in trial 1 and 2 (Table 6 and Table 7) and at Midwest Laboratories

(Omaha, NE, USA) for proximate and mineral composition in trial 3(Table 8).

99

2.2. Growth trials

Three trials were conducted at the E.W. Shell Fisheries Research Station, Auburn

University (Auburn, AL, USA). Pacific white shrimp post larvae (PL) were obtained from

Shrimp Improvement Systems (Islamorada, Florida) and nursed in an indoor recirculating

system. PLs were fed a commercial feed (Zeigler Bros., Inc., Gardners, Pennsylvania, USA)

using an automatic feeder for ~1 week, and then switched to crumbled commercial shrimp feed

(Zeigler Bros., Inc., Gardners, Pennsylvania, USA) for ~1- 2 weeks.

In trial 1, the recirculating system consisted of 12 aquaria (160 L) connected to a

common reservoir, biological filter, bead filter, fluidized biological filter and recirculation pump.

Four replicate groups of shrimp (1.51 g initial mean weight; 8 shrimp / tank) were offered diets

using our standard feeding protocol over 6 weeks. Based on historic results, feed inputs were

pre-programmed assuming the shrimp would double their weight weekly up to one gram then

gain 0.8-1.3 g weekly with a feed conversion ratio (FCR) of 1.8. Daily allowances of feed were

adjusted based on observed feed consumption, weekly counts of the shrimp and mortality.

Consequently, for each tank in trial 1, a fixed ration of 1.65 g day-1 for the first and second week,

1.85 g day-1 for the third week, 2.26 g day-1 for the fourth week, 2.47 g day-1 for the fifth week,

and 2.67 g day-1 for the remaining culturing period was offered, partitioned in 4 feedings each

day.

In trial 2 and trial 3, the recirculating system consisted of 24 aquaria (135 L) connected to

a common reservoir, biological filter, bead filter, fluidized biological filter and recirculation

pump. Four replicate groups of shrimp (In trial 2: 0.98 g initial mean weight, 10 shrimp / tank; In

trial 3: 0.15 g initial mean weight, 10 shrimp / tank) were offered diets using our standard

feeding protocol over 6 weeks. Based on historic results, feed inputs were pre-programmed

100

assuming the shrimp would double their weight weekly up to one gram then gain 0.8-1.3 g

weekly with a feed conversion ratio (FCR) of 1.8. Daily allowances of feed were adjusted based

on observed feed consumption, weekly counts of the shrimp and mortality. Consequently, for

each tank in trial 2, a fixed ration of 2.06 g day-1 for the first week, 2.31 g day-1 for the second

week, 2.57 g day-1 for the third week, 2.83 g day-1 for the fourth week, 3.09 g day-1 for the fifth

week, and 3.34 g day-1 for the last week was allotted in 4 portions per day. For each tank in trial

3, a fixed ration of 0.39 g day-1 for the first week, 0.83 g day-1 for the second week, 1.66 g day-1

for the third week, 2.24 g day-1 for the fourth week, 2.53 g day-1 for the fifth week, and 3.05 g

day-1 for the last week was also allotted in 4 portions per day.

At the conclusion of each growth trial, shrimp were counted and group-weighted. Mean

final weight, FCR, WG, biomass, and survival were determined (Table 9, Table 10, and Table

11). After obtaining the final total weight of shrimps in each aquarium, 4 shrimps from trial 2

and trial 3 were randomly selected and frozen at -20 °C for subsequent determination of whole

body composition. Proximate composition and amino acid profile (Table 12 and Table 14) of

whole shrimp was analyzed by University of Missouri-Columbia, Agriculture Experiment

Station Chemical Laboratory (Columbia, MO, USA). Protein and amino acids retention

efficiencies were calculated as follows:

Protein retention (%) = (final weight × final protein content) - (initial weight × initial protein

content) × 100 / protein offered.

Amino acids (AA) retention (%) = (final weight × final AA content) - (initial weight × initial AA

content) × 100 / AA offered.

101

2.3. Water quality monitoring

For all trials, dissolved oxygen (DO), water temperature and salinity were measured

twice daily by using a YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA).

Hydrogen potential (pH) was measured twice weekly by using a waterproof pHTestr30 (Oakton

instrument, Vernon Hills, IL, USA). Total ammonia-nitrogen (TAN) and nitrite were evaluated

every week by using the methods described by Sororzano (1969) and Spotte (1979).

2.4. Digestibility trial

The digestibility trial was conducted in the mentioned recirculation system and utilized

six shrimp per aquaria with six aquaria per dietary treatment. Once acclimated for three days to

the test diets, feces from two aquaria were pooled (n=3) and collected over a five-day period or

until adequate samples were obtained. To obtain fecal samples, the aquaria were cleaned by

siphoning before each feeding with the first collection of the day discarded. After cleaning, the

shrimp were offered an excess of feed and then about 1 hour later feed was removed and feces

were collected by siphoning onto a 500 µm mesh screen. Collected feces were rinsed with

distilled water, dried at 105 °C until a constant weight was obtained, and then stored in freezer

(−20 °C) until analyzed. Apparent digestibility coefficient for dry matter, protein, energy, and

amino acids were determined by using chromic oxide (Cr2O3, 1%) as an inert marker. Chromium

concentrations were determined by the method of McGinnis and Kasting (1964) in which, after a

colorimetric reaction, absorbance is read on a spectrophotometer (Spectronic genesis 5, Milton

Roy Co., Rochester, NY, USA) at 540nm. Gross energy of diets and fecal samples were analyzed

with a Semi micro-bomb calorimeter (Model 1425, Parr Instrument Co., Moline, IL, USA).

Protein were determined by micro-Kjeldahl analysis (Ma and Zuazaga, 1942). Amino acids were

102

analyzed by University of Missouri-Columbia, Agriculture Experiment Station Chemical

Laboratory (Columbia, MO, USA). The apparent digestibility coefficient of dry matter (ADMD),

protein (APD), energy (AED), and amino acids (AAAD) were calculated according to Cho et al.

(1982) as follows:

ADMD (%) = 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces)]

APD, AED, and AAAD (%) = 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces) × (% nutrient

in feces / % nutrient in feed)

The apparent digestibility coefficients (ADC) of the test ingredients for dry matter,

energy, protein and amino acids were calculated according to Bureau and Hua (2006) as follows:

ADCtest ingredient = ADCtest diet + [(ADCtest diet – ADCref. diet) × (0.7 × Dref / 0.3 × Dingr)]

where Dref = % nutrient (or KJ/g gross energy) of reference diet mash (dry weight); Dingr = %

nutrient (or KJ/g gross energy) of test ingredient (dry weight).

2.5. Statistical analysis

All the data were analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). Data from

three growth trials were analyzed using one-way ANOVA to determine significant differences

(P<0.05) among treatments followed by the Tukey’s multiple comparison test to determine

difference between treatments in each trial. The pooled standard errors were used across growth

trials, as the variance of each treatment is the same. Arcsine square root transformation was used

prior to analysis for the proportion data. False discover rate (FDR) controlling procedures were

used to adjust the P-value to control the FDR for data from nutrient contents of whole body and

amino acid retention. Data from digestibility trial were analyzed using non-parametric (kruskal-

103

wallis) one-way ANOVA to determine significant differences (P<0.05) among treatments

followed by the Tukey’s multiple comparison test to determine differences between treatments.

3. Results

3.1. Water quality

In trial 1, DO, temperature, salinity, pH, TAN, and nitrite were maintained at 6.15 ± 0.49

mg L-1, 28.1 ± 1.6 °C, 9.6 ± 0.7 ppt, 7.4 ± 0.3, 0.17 ± 0.05mg L-1, and 0.16 ± 0.07 mg L-1,

respectively. In trial 2, DO, temperature, salinity, pH, TAN, and nitrite were maintained at 6.20 ±

0.73 mg L-1, 29.5 ± 0.9 °C, 8.4 ± 1.0 ppt, 7.5 ± 0.3, 0.09 ± 0.10 mg L-1, and 0.05 ± 0.04 mg L-1,

respectively. In trial 3, temperature, salinity, pH, TAN, and nitrite were maintained at 6.96 ±

0.31 mg L-1, 28.1 ± 0.3 °C, 8.2 ± 0.6 ppt, 7.0 ± 0.3, 0.05 ± 0.04 mg L-1, and 0.12 ± 0.12 mg L-1,

respectively. Water quality conditions in all three trials were suitable for normal growth and

survival of this species.

3.2. Growth trials

Performances of Pacific white shrimp offered diets with various BB levels in trial 1, 2

and 3 are presented in Table 9, 10, and 11, respectively. In trial 1, shrimp fed with diets

incorporated with BB exhibited significantly improved survival. However, final mean weight

and WG were significantly reduced when shrimp fed with diets contained 12% BB. Whereas

FCR was significantly increased when shrimp fed with diets supplemented with both 6 and 12%

BB. No significant difference was observed in final biomass (45.70 to 53.85 g) across the

treatments

104

In trial 2, final biomass was significantly reduced when 12% BB was included in the

practical shrimp diet. Significant improvements in final mean weight and WG whereas

dramatically reduced FCR were determined when shrimp fed with the diet concluded 1% BB

compared to those fed with diet supplemented with 6 and 12% BB. No significant difference was

found in survival (92.5 to 100%) across all the treatments.

In trial 3, final biomass was significantly reduced when 26.6% BB was added in the diet

compared to the diet without BB supplementation. Shrimp fed with diets contained 12% and

26.6% BB exhibited significantly lower WG than those fed with diet did not contain BB.

Significant increment in FCR was determined in the diet incorporated with 12 and 26.6% BB

compared to the treatment supplemented with 0 and 13.3% BB. No significant difference was

detected in survival (90 to 100%) across all the treatments.

3.3. Whole body composition

Proximate composition and amino acids profile of whole shrimp body in trial 2 and trial 3

are presented in Table 12 and Table 14. Dietary BB inclusion at 12% significantly improved

protein content while decreased lipid content of whole shrimp body. Moisture content of shrimp

in the treatment fed with 12% BB was significantly lower than the treatment contained 4% BB.

Arginine and glycine contents were significantly improved when BB was supplemented at 12%

than other treatments. Significant improvement was detected, whereas reduction was also

observed in the diet contained 12% BB compared to the treatment supplemented with 0, 1, and

2% BB. Threonine content of shrimp body fed with diet contained 1% BB was significantly

higher than other treatments. No significant differences were detected in alanine, aspartic acid,

cysteine, glutamic acid, histidine, hydroxylysine, hydroxyproline, isoleucine, leucine, lysine,

105

phenylalanine, serine, tryptophan, tyrosine, and valine concentrations of whole shrimp body

across all the treatments.

In trial 3, protein and ash content of shrimp fed with diet contained 26.6% BB was

significantly improved compared to those fed with diet supplemented with 0 and 6% BB. Lipid

content of shrimp fed with diet included with 13.3 and 26.6% BB was dramatically reduced in

contrast with those offered with diet contained 0 and 6% BB. No significant effects were

detected in moisture (76.1 to 77.4%) and fiber (5.25 to 5.68%) contents.

3.4. Protein and amino acid retentions

Protein and amino acids retention efficiencies of Pacific white shrimp in trial 2 and trial 3

are shown in Table 13 and Table 14. In trial 2, PRE was significantly reduced when shrimp fed

with diets contained 12% BB compared to other treatments. There were reasonable

correspondences of total AAs and individual AAs retention efficiencies to PRE. In general, total

AA and most of individual AAs retention efficiencies except hydroxylysine, hydroxyproline, and

tyrosine retention efficiency were significantly depressed when shrimp fed with diet contained

12% BB compared to other treatments. Hydroxyproline retention efficiency was significantly

higher in the diet incorporated with 4% BB than other treatments. Tyrosine retention efficiency

was significantly reduced in the treatment supplemented with 12% BB compared to the

treatments contained 0, 1, 2, and 4% BB. No significant difference was detected in the multiple

comparison of hydroxylysine, which may due to the variance. In trial 3, PRE was significantly

depressed when 26.6% BB was incorporated in the diet.

106

3.5. Digestibility trial

ADMD, APD, and AED for the diet (D) and ingredient (I) using 70:30 replacement

technique offered to Pacific white shrimp are presented in Table 15. The digestibility trial

contained a range of ingredients; hence, we have provided a few other ingredients as a reference.

To confirm the digestibility results, faecal samples for basal diet, FM diet, and BB diet were

recollected. Basal1 and Basal2, FM1 and FM2, and BB1 and BB2 represent first collection and

second collection of basal diet, FM diet, and BB diet, respectively. ADMD, AED, and APD of

BB were significantly lower than those of FM and SBM.

AAAD values for the SBM, FM and BB using 70:30 replacement technique offered to

Pacific white shrimp are presented in Table 16. Apparent digestibility coefficients of alanine,

arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysing,

methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and total

amino acids of BB were significantly lower than those of FM and SBM. Total AA and individual

AA digestibility coefficients were reasonably corresponded to APD.

4. Discussion

The nutrient digestibility of a feed ingredient is an important factor to evaluate the overall

nutritive value of the ingredient because it is related to the quantity of the nutrient absorbed by

the animals. SBM had the highest APD (97.03%), AED (82.56%), and AAAD (90.78 – 96.91%)

among the ingredients tested in the current study. Similar ranges of results for APD, AED, and

AAAD were reported in multiple shrimp studies (Cruz-Suárez et al., 2009; Fang et al., 2016; Liu

et al., 2013; Yang et al., 2009; Zhou et al., 2015). APD and AED of FM1 were 67.07% and

69.77%, respectively. Similar results were acquired in FM2 (APD and AED: 71.3% and 65.78%,

107

respectively). The analogous results of basal diet and FM diet from the collections under two

occasions pointed to the consistency in the feces collection and sample analysis methods. APD

of FM has been reported to be ranged from 62.7% to 91.6% in numerous studies (Lemos et al.,

2009; Liu et al., 2013; Terrazas-Fierro et al., 2010; Yang et al., 2009). ADP of FM in our study

were in the lower range of the results documented among those researches. The differences in

digestibility of FM among various studies could be attributed to several factors, such as different

raw materials, location or processing methods used to produce the products, and unknown

factors related to different production batches.

Physiologically, ADCs should not below 0% or above 100%. However, ADMD of BB1

was negative, and the same phenomenon was confirmed in the recollected faecal sample (BB2).

The negative ADMD resulted in very low values of APD, AED, and AAAD of BB for Pacific

white shrimp. Similarly, Akiyama et al. (1989) also documented negative ADMD values of some

dietary fillers (diatomaceous sand, chitin, and cellulose) for Pacific white shrimp. Unexpected

negative ADMD value may be attributed to an unidentified interaction among the ingredients of

the feed or endogenous losses by the animal from processes such an enzymatic secretion,

sloughing of gut epithelial cells, formation of the chitinous peritrophic membrane, and the

secretion of other lubricative substances (Akiyama et al., 1989). Another explanation of the

negative ADMD value may be due to the leaching of chromic oxide of the feed before

consumption or from the fecal samples. However, in the present study leaching of chromic oxide

would be negligible for short exposure time to the seawater. Hence, the negative ADMD may

indicate that BB cannot be digested by Pacific white shrimp. Ingredient digestibility data of BB

for shrimp is still limited. Two digestibility studies of BB for rainbow trout and Atlantic salmon

were available (Øverland et al., 2006; Storebakken et al., 1998). However, neither of these

108

researches documented negative values, all the digestibility values they reported were in the

normal ranges. Differences among these studies may be attributed to different aquatic animals

and bacterial proteins.

Using methane-oxidising bacteria as protein and amino acid sources in aquatic animal

nutrition recently received considerable attention due to the abundant supply, cheap

transportation, and reasonable cost of natural gas (Øverland et al., 2010). In the current study,

significantly reduced growth performance was detected when the diet was supplemented with 12%

BB in trial 1. FCR was significantly increased when the diets were incorporated with both 6 and

12% BB. To demonstrate the results from trial 1 and explore the effects of BB supplementation

at low levels, the basal diet was incorporated with 0, 1, 2, 4, 6, and 12% BB in trial 2. Our

findings indicated that no significant differences were observed in terms of growth performance

and FCR when the diets supplemented with BB up to 6%. However, dietary BB incorporation at

12% dramatically reduced the WG and increased FCR, which were in accordance with the

results documented in trial 1. Similarly, Aas et al. (2007) indicated that bacterial protein meal

(BPM) can be used up to 9% in the diets for Atlantic halibut but supplementation of BPM at 18%

caused depression in growth. By contrast, Aas et al. (2006a) reported that BPM can be utilized

up to 36% in the diets for Atlantic salmon without compromising the growth. Similar results

were demonstrated by Berge et al. (2005) and Storebakken et al. (2004) who documented that

BPM can be supplemented in Atlantic salmon diets at 20% and 19.3%, respectively. The

inclusions of BPM up to 27% were also confirmed to be successfully applied in the diets for

rainbow trout (Aas et al., 2006b). Factors caused the inconsistent results with supplemental BB

may be different strains utilized to produce BB and various aquatic animal species investigated

among the experiments.

109

To elucidate if the digestible protein is the cause of the depressed growth and increased

FCR, another growth trial (trial 3) was initiated to essentially use the same diets that contained 0,

6 and 12% BB in trial 2. Additionally, two more diets were formulated to replace the same ratio

of SBM as diets contained 6 and 12% BB, respectively, but on a digestible protein basis. Dietary

12% BB supplementation significantly reduced WG but increased FCR in trial 3, which has been

proven in both trial 1 and trial 2. The diets balanced on digestible protein basis performed

essentially the same as those did not balanced for digestible protein in terms of WG and FCR,

which may reveal that BB evaluated in the current study is not digestible by shrimp or the

digestibility of this product is still in question.

With regards to the proximate composition of whole shrimp body, lipid level was

dramatically reduced, while protein content was significantly enhanced when shrimp fed with

diet contained 12% BB in trial 2. Similar trends of protein and lipid content of whole body were

determined in trial 3. In trial 3, shrimp fed with diets contained 13.3 and 26.6% BB exhibited

significantly improved protein and depressed lipid contents. By contrast, no significant effects of

dietary BB supplementation on the protein and lipid content of fish body were detected in many

other studies (Aas et al. 2006a; b; 2007; Berge et al., 2005, Storebakken et al., 2004). The

significantly reduced lipid content of shrimp body in trial 2 and trial 3 may be caused by low

digestible energy in the diet contained 12% BB as this ingredient had significantly lower energy

digestibility than SBM. The improvements in protein content of whole body in trial 2 and trial 3

might be indirectly response to the decreased lipid content.

In the present study, PRE was significantly reduced when the diet was supplemented with

12% BB, and there was a reasonable correspondence to the total AA and individual AA amino

acids retention efficiency in trial 2. In general, total AA and most of individual AAs except

110

hydroxylysine, hydroxyproline, and tyrosine retention efficiencies were significantly depressed

when shrimp fed with diet contained 12% BB. Similarly, Aas et al. (2006b) reported that

significantly reductions were determined in PRE and most individual AA retention efficiency

when rainbow trout was fed with diet supplemented with 18% or 27% BB. In addition, Aas et al.

(2007) documented that PRE and indispensable AA retention efficiency were significantly

decreased when BB was supplemented at 18% in Atlantic halibut diet. PRE was determined by a

number of factors including dietary protein levels, feed intake, final weight and initial weight of

animals as well as the final and initial protein content of animals (Halver and Hardy, 2002). In

trial 2, no significant differences were detected in dietary protein levels, feed offered to the

shrimp, and initial weight of shrimp. Although protein content of whole shrimp body was

significantly improved in the diet contained 12% BB, its effect cannot counteract with the

significantly reduced final mean weight of shrimp in the same treatment, which is the primary

cause of reduced PRE. In trial 3, a similar decreasing trend was observed when BB was included

at 12% and 26.6%.

Survival of shrimp was significantly enhanced in trial 1 when diets were incorporated

with both 6 and 12% BB. However, no significant differences were observed in survival across

all the treatments in trial 2 and 3. Similarly, no mortality problems were reported in multiple

researches investigated BB as protein sources in different kinds of fish (Aas et al., 2006a; b;

2007; Berge et al., 2005; Storebakken et al., 2004). Survival is not repeatable as it is impossible

to conduct experiments under the same conditions in all three trials. The significantly improved

survival by BB supplementation in trial 1 indicated that BB might induce immune responses in

shrimp. Hence, further study considering the immune effects of BB supplementation at low

levels is warranted.

111

5. Conclusion

Under the conditions of the present study BB can be utilized up to 4% in shrimp feed

without causing growth depression. However, supplementations (≥ 6%) of BB can result in

negative effects on growth response, FCR, and protein as well as amino acids retention

efficiency. Given the negative result of dry matter digestibility of BB and no improvements in

the treatments balanced on digestible protein basis by using BB, we may infer that the negative

effects caused by high incorporation levels of BB may due to low nutrient digestibility of this

ingredient for shrimp. Based on dramatically enhanced survival in the treatment with BB

supplementation in trial 1, further research regarding the immune effects low inclusion levels of

BB in practical shrimp feed is warranted.

112

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116

Table 1 Composition (% as is) of test diets utilized in trial 1.

Ingredient Diet code

T1D1 T1D2 T1D3 Soybean meal1 54.10 47.40 40.50 Corn protein concentrate2 8.00 8.00 8.00 Whole wheat3

25.00 25.00 25.00 Bacterial biomass4

0.00 6.00 12.00 Fish oil2 6.05 6.14 6.24 Trace mineral premix5 0.50 0.50 0.50 Vitamin premix6 1.80 1.80 1.80 Choline chloride3 0.20 0.20 0.20 Stay C7 0.10 0.10 0.10 Mono-dicalcium phosphate8 2.50 2.50 2.50 Lecithin9 1.00 1.00 1.00 Cholesterol3 0.05 0.05 0.05 Corn starch3 0.70 1.31 2.11 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 3 MP Biomedicals Inc., Solon, OH, USA. 4 KnipBio Inc., Lowell, MA, USA. 5 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA.

117

Table 2 Composition (% as is) of test diets utilized in trial 2.

Ingredient Diet code

T2D1 T2D2 T2D3 T2D4 T2D5 T2D6 Fish meal1 6.00 6.00 6.00 6.00 6.00 6.00 Soybean meal2 53.00 51.90 50.80 48.60 46.50 40.10 Corn protein concentrate3 8.00 8.00 8.00 8.00 8.00 8.00 Bacteria biomass4

0.00 1.00 2.00 4.00 6.00 12.00 Fish oil2 5.92 5.93 5.94 5.95 5.97 6.01 Trace mineral premix6 0.50 0.50 0.50 0.50 0.50 0.50 Vitamin premix7 1.80 1.80 1.80 1.80 1.80 1.80 Choline chloride5 0.20 0.20 0.20 0.20 0.20 0.20 Stay C8 0.10 0.10 0.10 0.10 0.10 0.10 Mono-dicalcium phosphate9 2.50 2.50 2.60 2.60 2.80 2.90 Lecithin10 1.00 1.00 1.00 1.00 1.00 1.00 Cholesterol5 0.08 0.08 0.08 0.08 0.08 0.08 Methionine11

0.05 0.05 0.04 0.04 0.04 0.02 Lysine11

0.00 0.01 0.01 0.03 0.04 0.07 Corn starch5 20.85 20.93 20.93 21.10 20.97 21.22 1 Omega Protein Inc., Huston TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 KnipBio Inc., Lowell, MA, USA. 5 MP Biomedicals Inc., Solon, OH, USA. 6 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 7 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 8 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 9 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 10 The Solae Company, St. Louis, MO, USA. 11 Aldrich-Sigma, St. Louis, MO, USA.

118

Table 3 Composition (% as is) of test diets utilized in trial 3.

Ingredients Diet code

T3D1 T3D2 T3D3 T3D4 T3D5 Fish meal1 6.00 6.00 6.00 6.00 6.00 Soybean meal2 53.00 46.50 46.50 40.10 40.10 Corn protein concentrate3 8.00 8.00 8.00 8.00 8.00 Bacteria biomass4

0.00 6.00 13.30 12.00 26.60 Fish oil2 5.92 5.97 5.81 6.01 5.70 Trace mineral premix6 0.50 0.50 0.50 0.50 0.50 Vitamin premix7 1.80 1.80 1.80 1.80 1.80 Choline chloride5 0.20 0.20 0.20 0.20 0.20 Stay C8 0.10 0.10 0.10 0.10 0.10 Mono-dicalcium phosphate9 2.50 2.80 2.80 2.90 2.90 Lecithin10 1.00 1.00 1.00 1.00 1.00 Cholesterol5 0.08 0.08 0.08 0.08 0.08 Methionine11

0.05 0.04 0.03 0.02 0.02 Lysine11

0.00 0.04 0.05 0.07 0.09 Corn starch5 20.85 20.97 13.83 21.22 6.91 1 Omega Protein Inc., Huston TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 KnipBio Inc., Lowell, MA, USA. 5 MP Biomedicals Inc., Solon, OH, USA. 6 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 7 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 8 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 9 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 10 The Solae Company, St. Louis, MO, USA. 11 Aldrich-Sigma, St. Louis, MO, USA.

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Table 4 Composition of reference diet for the determination of digestibility coefficients of bacteria biomass (BB)

Ingredients % as is

Soybean meal1 10.00

Fish meal2 32.50

Fish oil2 3.20

Whole wheat3 47.60

Trace mineral premix4 0.50

Vitamin premix5 1.80

Choline cloride6 0.20

Stay C7 0.10

Corn starch3 1.00

Lecethin8 1.00

Chromic oxide9 1.00 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Omega Protein Inc., Houston TX, USA. 3 MP Biomedicals Inc., Solon, OH, USA 4 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 5 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 6 VWR, Radnor, PA, USA. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 The Solae Company, St. Louis, MO, USA. 9 Alfa Aesar, Haverhill, MA, USA.

120

Table 5 Proximate composition (% as is), phosphorus content (% as is), and amino acid profile (% as is) of the ingredients used in the growth and digestibility trials.

Composition1 Bacterial biomass Fish meal Soybean meal

Crude protein 52.42 62.78 44.89

Moisture 3.50 7.99 10.97

Crude fat 1.29 10.56 3.78

Crude fiber 0.00 0.00 3.20

Ash 4.25 18.75 6.67

Phosphorus 1.00 3.15 0.66

Alanine 3.81 3.91 2.04

Arginine 3.23 3.68 3.35

Aspartic acid 3.72 5.34 5.10

Cysteine 0.30 0.47 0.62

Glutamic acid 6.13 7.47 8.24

Glycine 2.73 4.88 2.04

Histidine 0.80 1.63 1.20

Isoleucine 1.80 2.42 2.17

Leucine 3.24 4.21 3.57

Lysine 2.79 4.67 3.06

Methionine 0.86 1.61 0.66

Phenylalanine 2.04 2.39 2.35

Proline 2.25 3.08 2.39

Serine 1.21 2.11 1.90

Taurine 0.08 0.73 0.13

Threonine 2.00 2.41 1.75

Tryptophan 0.10 0.62 0.62

Tyrosine 1.34 1.67 1.64

Valine 2.84 2.99 2.34 1 Ingredients were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

121

Table 6 Proximate composition (% as is) and amino acid profile (% as is) of the test diets used in trial 1.

Composition1 T1D1 T1D2 T1D3

Crude Protein 37.67 36.37 36.77 Moisture 5.41 8.34 6.66 Crude Fat 9.54 8.71 8.49 Crude Fiber 4.05 3.48 3.05 Ash 6.06 5.65 5.51 Alanine 1.90 1.92 2.01 Arginine 2.26 2.17 2.16 Aspartic Acid 3.51 3.26 3.16 Cysteine 0.55 0.52 0.49 Glutamic Acid 7.40 6.88 6.76 Glycine 1.48 1.46 1.50 Histidine 0.92 0.86 0.83 Isoleucine 1.69 1.59 1.56

Leucine 3.48 3.29 3.23 Lysine 1.92 1.83 1.81 Methionine 0.60 0.65 0.62 Phenylalanine 1.99 1.90 1.87 Proline 2.40 2.27 2.25 Serine 1.58 1.46 1.44 Taurine 0.10 0.10 0.10 Threonine 1.34 1.30 1.31 Tryptophan 0.39 0.40 0.36 Tyrosine 1.53 1.47 1.43 Valine 1.80 1.73 1.74 1 Diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

122

Table 7 Proximate composition (% as is) and amino acid profile (% as is) of the test diets used in trial 2. Composition1 T2D1 T2D2 T2D3 T2D4 T2D5 T2D6 Crude protein 36.33 35.52 36.42 34.29 34.48 36.10 Moisture 7.15 8.57 7.34 9.47 9.42 8.08 Crude fat 9.39 9.44 8.94 9.36 9.83 8.15 Crude fiber 3.21 3.28 3.01 2.99 2.73 2.69 Ash 6.86 6.75 6.70 6.62 6.60 6.55 Alanine 1.87 1.88 1.97 1.85 1.91 2.01 Arginine 2.18 2.14 2.14 2.09 2.10 2.08 Aspartic Acid 3.44 3.42 3.42 3.24 3.22 3.16 Cysteine 0.48 0.47 0.46 0.43 0.43 0.40 Glutamic Acid 6.33 6.26 6.39 5.93 5.96 5.88 Glycine 1.56 1.54 1.59 1.52 1.55 1.54 Histidine 0.86 0.85 0.86 0.80 0.80 0.78 Isoleucine 1.60 1.61 1.63 1.53 1.53 1.53 Leucine 3.28 3.25 3.37 3.09 3.14 3.21 Lysine 2.01 2.00 2.00 1.94 1.94 1.92 Methionine 0.64 0.63 0.68 0.61 0.60 0.58 Phenylalanine 1.85 1.84 1.87 1.75 1.76 1.77 Proline 2.13 2.04 2.14 2.00 2.04 2.07 Serine 1.48 1.43 1.47 1.37 1.39 1.37 Taurine 0.16 0.14 0.14 0.15 0.14 0.14 Threonine 1.29 1.28 1.30 1.24 1.26 1.27 Tryptophan 0.47 0.49 0.45 0.43 0.44 0.45 Tyrosine 1.33 1.25 1.27 1.26 1.25 1.23 Valine 1.73 1.75 1.78 1.67 1.70 1.80 1 Diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

123

Table 8 Proximate composition1 (% as is) and mineral composition1 (g kg-1: phosphorus, sulfur, potassium, magnesium, calcium, sodium; mg kg-1: iron, manganese, copper, zinc) of the test diets used in trial 3.

Composition1 T3D1 T3D2 T3D3 T3D4 T3D5 Crude protein 35.7 33.7 38.4 34.7 41.1

Moisture 8.7 11.71 8.39 9.7 10.46 Crude fat 6.71 7.57 8.2 7.64 7.26

Crude fiber 3.1 2.47 7.1 2.62 8.3 Ash 7.08 6.67 7.08 6.61 6.56

Sulfur 0.40 0.36 0.44 0.36 0.43 Phosphorus 1.36 1.36 1.29 1.47 1.37 Potassium 1.33 1.16 1.23 1.13 1.11

Magnesium 0.18 0.16 0.19 0.15 0.17 Calcium 1.31 1.27 1.37 1.25 1.37 Sodium 0.10 0.11 0.14 0.13 0.16

Iron (ppm) 149 127 166 126 161 Manganese (ppm) 40.1 38.1 67.8 43.4 69.4

Copper (ppm) 16.8 15.9 16.9 14.6 15.8 Zinc (ppm) 183 168 213 166 177

1 Diets were analyzed at Midwest Laboratories (Omaha, NE, USA).

124

Table 9 Performance of juvenile shrimp L. vannamei (Initial weight 1.51g) offered diets with different bacterial biomass levels (0, 6, and 12 %) for six weeks in trial 1.

Diet BB levels

(%)

Final

biomass (g)

Final mean

weight (g) WG3 (%) FCR2 Survival (%)

T1D1 0 49.25 8.26a 440.04a 1.65a 75.0b

T1D2 6 53.85 6.96ab 370.55ab 1.99b 96.9a

T1D3 12 45.70 5.72b 280.94b 2.61b 100.0a

PSE1 1.1211 0.1646 1.7274 0.0603 12.4145

P-value 0.0831 0.0014 0.0012 0.0010 0.0047 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - Initial weight) / Initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

125

Table 10 Performance of juvenile shrimp L. vannamei (Initial weight 0.98g) offered diets with different bacteria biomass levels (0, 1, 2, 4, 6, and 12 %) for six weeks in trial 2.

Diet BB levels (%) Final

biomass (g)

Final mean

weight (g) WG3 (%) FCR2 Survival

(%)

T2D1 0 79.3a 8.4ab 766.6ab 1.64bc 95.0

T2D2 1 84.9a 9.2a 836.8a 1.50c 92.5

T2D3 2 84.0a 8.6ab 811.1ab 1.56bc 97.5

T2D4 4 85.3a 8.5ab 765.3ab 1.63bc 100.0

T2D5 6 75.3a 7.7b 697.5b 1.83b 97.5

T2D6 12 58.1b 5.8c 493.70c 2.50a 100.0

PSE1 1.2191 0.1031 13.7258 0.0303 1.0623

P-value <0.0001 <0.0001 <0.0001 <0.0001 0.1458 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - Initial weight) / Initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

126

Table 11 Performance of juvenile shrimp L. vannamei (Initial weight 0.15g) offered diets formulated to partially replace soybean meal on a digestible protein basis for six weeks in trial 3.

Diet BB levels

(%)

Final

biomass (g)

Final mean

weight (g) WG3 (%) FCR2 Survival (%)

T3D1 0 42.68a 4.74a 3160.39a 1.72c 90.0

T3D2 6 43.15ab 4.30ab 2813.38ab 1.90bc 100.0

T3D3 13.3 45.38ab 4.54a 2732.16abc 1.73c 100.0

T3D4 12 38.48ab 3.84bc 2438.14bc 2.11ab 100.0

T3D5 26.6 35.05b 3.60c 2304.94c 2.26a 97.5

PSE1 1.1420 0.0710 57.1783 0.0338 1.9084

P-value 0.0406 0.0002 0.0008 0.0001 0.3194 1 PSE: Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - Initial weight) / Initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

12

7

Tab

le 1

2 Pr

oxim

ate

com

posi

tion2 (

moi

stur

e: %

as

is; p

rote

in a

nd li

pid:

% d

ry w

eigh

t) an

d am

ino

acid

pro

file2 (

% d

ry w

eigh

t) of

who

le sh

rimp

body

off

ered

die

ts w

ith d

iffer

ent b

acte

ria b

iom

ass l

evel

s (0,

1, 2

, 4, 6

, and

12

%) f

or si

x w

eeks

in tr

ial 2

.

Die

t T 2

D1

T 2D

2 T 2

D3

T 2D

4 T 2

D5

T 2D

6 PS

E1 P-

valu

e A

djus

t P-v

alue

B

B le

vels

(%)

0 1

2 4

6 12

M

oist

ure

75.6

5ab

75.4

8ab

75.7

9ab

75.1

7b 76

.93ab

77

.23a

0.20

91

0.01

17

0.03

51

Prot

ein

75.0

8b 74

.97b

74.3

9b 74

.80b

75.2

8b 77

.77a

0.18

07

<0.0

001

0.00

03

Lipi

d 6.

37b

6.54

ab

7.92

a 7.

26ab

5.

76b

3.62

c 0.

1706

<0

.000

1 <0

.000

1 A

lani

ne

4.27

4.

33

4.44

4.

28

4.40

4.

31

0.03

65

0.50

92

0.55

55

Arg

inin

e 5.

45bc

5.

23c

5.43

c 5.

47bc

5.

72b

6.17

a 0.

0325

<0

.000

1 <0

.000

1 A

spar

tic A

cid

6.76

6.

94

6.79

6.

71

6.84

6.

86

0.03

22

0.21

40

0.34

25

Cys

tein

e 0.

60

0.61

0.

61

0.60

0.

62

0.63

0.

0039

0.

0673

0.

1614

G

luta

mic

Aci

d 10

.18

10.4

1 10

.16

10.0

6 10

.22

10.3

1 0.

0559

0.

3439

0.

4855

G

lyci

ne

5.01

cd

4.83

d 5.

01cd

5.

19bc

5.

49b

6.08

a 0.

0350

<0

.000

1 <0

.000

1 H

istid

ine

1.49

1.

52

1.49

1.

48

1.50

1.

52

0.01

15

0.68

61

0.71

59

Hyd

roxy

lysi

ne

0.14

0.

15

0.17

0.

18

0.17

0.

17

0.00

70

0.50

32

0.55

55

Hyd

roxy

prol

ine

0.20

0.

22

0.23

0.

21

0.21

0.

20

0.00

51

0.49

81

0.55

55

Isol

euci

ne

2.95

3.

00

2.95

2.

93

2.97

2.

95

0.01

11

0.38

29

0.51

05

Leuc

ine

4.95

5.

03

4.94

4.

92

4.97

5.

01

0.01

62

0.18

96

0.32

50

Lysi

ne

4.92

5.

04

4.98

4.

93

5.04

5.

11

0.02

39

0.08

74

0.19

07

Met

hion

ine

1.46

c 1.

49c

1.50

bc

1.51

abc

1.55

ab

1.57

a 0.

0065

0.

0002

0.

0010

Ph

enyl

alan

ine

3.16

3.

23

3.20

3.

18

3.20

3.

27

0.01

69

0.33

98

0.48

55

Prol

ine

4.09

a 4.

15a

4.18

a 4.

00ab

3.

88ab

3.

67b

0.04

15

0.00

36

0.01

43

Serin

e 2.

35

2.38

2.

35

2.34

2.

36

2.36

0.

0195

0.

9934

0.

9934

Th

reon

ine

2.62

b 2.

76a

2.62

b 2.

60b

2.64

b 2.

61b

0.01

35

0.00

49

0.01

69

12

8

Tryp

toph

an

0.87

0.

85

0.85

0.

85

0.85

0.

88

0.00

48

0.11

32

0.22

63

Tyro

sine

2.

51

2.33

2.

49

2.52

2.

45

2.57

0.

0405

0.

4536

0.

5555

V

alin

e 4.

10

4.19

4.

12

4.16

4.

17

4.00

0.

0257

0.

1632

0.

3014

To

tal

68.0

5 68

.66

68.4

9 68

.08

69.2

3 70

.23

0.25

81

0.06

29

0.16

14

1 PS

E: P

ool s

tand

ard

erro

r. 2 Pr

oxim

ate

com

posi

tion

and

amin

o ac

id p

rofil

e of

who

le b

ody

sam

ples

wer

e an

alyz

ed a

t Uni

vers

ity o

f Mis

sour

i-Col

umbi

a,

Agr

icul

ture

Exp

erim

ent S

tatio

n C

hem

ical

Lab

orat

ory

(Col

umbi

a, M

O, U

SA).

Val

ues w

ithin

a ro

w w

ith d

iffer

ent s

uper

scrip

ts a

re si

gnifi

cant

ly d

iffer

ent b

ased

on

Tuke

y’s m

ultip

le ra

nge

test

.

12

9

Tab

le 1

3 Pr

otei

n2 and

am

ino

acid

s3 rete

ntio

n ef

ficie

ncie

s of P

acifi

c w

hite

shrim

p at

the

conc

lusi

on o

f a 6

-wee

k gr

owth

tria

l 2 in

whi

ch

shrim

p w

ere

offe

red

diet

s for

mul

ated

to c

onta

in d

iffer

ent b

acte

ria b

iom

ass l

evel

s (0,

1, 2

, 4, 6

, and

12

%).

Ret

entio

n ef

ficie

ncie

s T 2

D1

T 2D

2 T 2

D3

T 2D

4 T 2

D5

T 2D

6 PS

E1 P-

valu

e A

djus

t P-v

alue

B

B le

vels

(%)

0 1

2 4

6 12

Pr

otei

n 34

.2ab

39

.0a

35.2

ab

38.3

a 31

.6b

22.1

c 0.

6018

<0

.000

1 <0

.000

1 A

lani

ne

37.5

ab

42.4

a 38

.9a

40.4

a 33

.2b

21.7

c 0.

5532

<0

.000

1 <0

.000

1 A

rgin

ine

41.5

a 45

.1a

43.9

a 46

.1a

39.8

a 31

.1b

0.76

23

<0.0

001

<0.0

001

Asp

artic

Aci

d 32

.6ab

37

.6a

34.4

ab

36.4

a 30

.8b

22.3

c 0.

5675

<0

.000

1 <0

.000

1 C

yste

ine

20.5

b 24

.0ab

22

.9ab

24

.6a

20.9

b 16

.3c

0.38

68

<0.0

001

<0.0

001

Glu

tam

ic A

cid

26.7

ab

30.9

a 27

.5ab

29

.9a

24.9

b 18

.0c

0.47

45

<0.0

001

<0.0

001

Gly

cine

53

.4ab

58

.0ab

54

.7ab

60

.6a

52.1

b 42

.1c

0.86

45

<0.0

001

<0.0

001

His

tidin

e 28

.6ab

33

.2a

30.0

ab

32.4

ab

27.1

b 19

.9c

0.59

00

<0.0

001

<0.0

001

Hyd

roxy

lysi

ne

29.3

40

.4

36.5

39

.4

31.2

22

.5

2.02

46

0.04

45

0.04

45

Hyd

roxy

prol

ine

16.9

b 18

.3b

21.1

b 36

.3a

11.9

c 9.

2c 0.

5395

<0

.000

1 <0

.000

1 Is

oleu

cine

30

.5ab

34

.5a

31.3

ab

33.7

a 28

.2b

19.8

c 0.

5290

<0

.000

1 <0

.000

1 Le

ucin

e 25

.0ab

28

.7a

25.4

ab

28.1

a 23

.0b

16.1

c 0.

4198

<0

.000

1 <0

.000

1 Ly

sine

40

.7bc

46

.9a

43.3

abc

45.0

ab

38.0

c 27

.6d

0.63

48

<0.0

001

<0.0

001

Met

hion

ine

38.1

b 43

.9a

38.5

ab

43.8

a 38

.0b

28.2

c 0.

6263

<0

.000

1 <0

.000

1 Ph

enyl

alan

ine

28.3

ab

32.5

a 29

.7ab

32

.1a

26.4

b 19

.0c

0.52

53

<0.0

001

<0.0

001

Prol

ine

32.4

bc

38.4

a 34

.5ab

35

.9ab

28

.0c

18.4

d 0.

6150

<0

.000

1 <0

.000

1 Se

rine

26.3

bc

30.8

a 27

.7ab

c 30

.1ab

24

.6c

17.6

d 0.

4686

<0

.000

1 <0

.000

1 Th

reon

ine

33.6

bc

40.1

a 34

.9bc

36

.8ab

30

.3c

21.1

d 0.

5590

<0

.000

1 <0

.000

1 Tr

ypto

phan

30

.5ab

31

.9ab

32

.3ab

34

.6a

27.9

b 20

.1c

0.58

20

<0.0

001

<0.0

001

Tyro

sine

31

.2a

34.3

a 34

.0a

35.2

a 28

.3ab

21

.4b

0.90

20

0.00

03

0.00

03

13

0

Val

ine

39.4

ab

44.5

a 40

.1ab

43

.9a

35.7

b 22

.7c

0.68

02

<0.0

001

<0.0

001

Tota

l 32

.3ab

36

.9a

33.7

ab

36.3

a 30

.1b

21.7

c 0.

5415

<0

.000

1 <0

.000

1 1 P

oole

d st

anda

rd e

rror

. 2 Pr

otei

n re

tent

ion

(%) =

(Fin

al w

eigh

t × F

inal

pro

tein

con

tent

) - (I

nitia

l wei

ght ×

Initi

al p

rote

in c

onte

nt) ×

100

/ Pr

otei

n of

fere

d.

3 A

min

o ac

ids (

AA

) ret

entio

n (%

) = (F

inal

wei

ght ×

Fin

al A

A c

onte

nt) -

(Ini

tial w

eigh

t × In

itial

AA

con

tent

) × 1

00 /

AA

off

ered

. V

alue

s with

in a

row

with

diff

eren

t sup

ersc

ripts

are

sign

ifica

ntly

diff

eren

t bas

ed o

n Tu

key’

s mul

tiple

rang

e te

st.

13

1

Tab

le 1

4 Pr

oxim

ate

com

posi

tion

of w

hole

shr

imp

body

and

pro

tein

ret

entio

n ef

ficie

ncy

(PR

E) o

ffer

ed d

iets

for

mul

ated

to

utili

ze

bact

eria

l bio

mas

s (B

B) p

artia

lly re

plac

e so

ybea

n m

eal o

n a

dige

stib

le p

rote

in b

asis

for s

ix w

eeks

in tr

ial 3

.

Die

t B

B le

vels

(%)

Prot

ein2 (%

) M

oist

ure

(%)

Lipi

d2 (%)

Fibe

r2 (%)

Ash

2 (%)

PRE3 (%

)

T 3D

1 0

70.8

3b 76

.1

8.40

a 5.

25

11.5

0c 30

.50a

T 3D

2 6

70.6

8b 76

.7

7.89

a 5.

26

11.8

0c 29

.37a

T 3D

3 13

.3

72.5

2ab

76.6

5.

07b

5.30

12

.56bc

27

.97a

T 3D

4 12

72

.59ab

77

.0

6.00

ab

5.48

13

.35ab

25

.43ab

T 3D

5 26

.6

73.5

5a 77

.4

4.07

b 5.

68

14.0

1a 20

.11b

P-va

lue

0.01

07

0.23

79

0.00

03

0.36

23

<0.0

001

0.00

02

PSE1

0.28

19

0.19

32

0.28

03

0.08

49

0.13

99

0.62

34

1 PS

E: P

ool s

tand

ard

erro

r. 2 D

ry w

eigh

t bas

is.

3 Pr

otei

n re

tent

ion

(%) =

(Fin

al w

eigh

t × F

inal

pro

tein

con

tent

) - (I

nitia

l wei

ght ×

Initi

al p

rote

in c

onte

nt) ×

100

/ Pr

otei

n of

fere

d.

13

2

Tab

le 1

5 A

ppar

ent d

ry m

atte

r (A

DM

), ap

pare

nt e

nerg

y (A

ED)

and

appa

rent

pro

tein

(A

PD)

dige

stib

ility

val

ues

for

the

diet

(D

) an

d in

gred

ient

(I) u

sing

70:

30 re

plac

emen

t tec

hniq

ue o

ffer

ed to

Pac

ific

whi

te sh

rimp

L. v

anna

mei

.

A

DM

DD

A

EDD

A

PDD

A

DM

DI

AED

I A

PDI

Bas

al1

76.3

8 ±

0.37

a 82

.66

± 1.

20a

92.0

8 ±

0.55

a

SB

M

77.0

2 ±

0.87

a 82

.63

± 1.

05ab

94

.76

± 0.

49a

78.5

1 ±

2.89

a 82

.56

± 3.

79a

97.0

3 ±

0.83

a

FM1

68.2

1 ±

3.80

b 78

.31

± 3.

21bc

80

.86

± 1.

80b

49.1

5 ±

12.6

7b 69

.77

± 9.

51ab

67

.07

± 4.

02b

BB

1 41

.12

± 2.

03c

59.8

6 ±

1.25

d 73

.09

± 1.

50c

-40.

11 ±

6.7

8c 13

.33

± 3.

85c

42.5

5 ±

3.92

c

Bas

al2

75.6

9 ±0

.52a

81.5

1 ±

0.41

ab

92.0

4 ±

0.03

a

FM2

67.9

9 ±

0.17

b 76

.44

± 0.

78c

82.3

4 ±

0.31

b 49

.45

± 0.

56ab

65

.78

± 2.

23b

71.3

0 ±

0.68

ab

BB

2 38

.54

± 1.

49c

53.9

8 ±

0.27

e 67

.48

± 1.

48d

-48.

16 ±

4.9

6c -0

.94

± 0.

82c

29.2

6 ±

3.79

c

Val

ues

from

eac

h tre

atm

ent a

re m

eans

and

sta

ndar

d de

viat

ion

of tr

iplic

ate

tank

s. V

alue

s w

ithin

a c

olum

n di

ffer

ent s

uper

scrip

ts a

re

sign

ifica

ntly

diff

eren

t (P

< 0.

05).

133

Table 16 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal (FM), bacteria biomass (BB) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei.

AA digestibility

coefficients (%) SBM FM BB

Alanine 93.75 ± 2.02a 69.09 ± 4.09b 51.05 ± 3.55c

Arginine 96.91 ± 1.44a 75.35 ± 3.78b 51.33 ± 3.43c

Aspartic acid 95.39 ± 1.36a 69.23 ± 3.70b 33.82 ± 4.79c

Cysteine 91.29 ± 1.68a 54.39 ± 7.06b -50.61 ± 16.31c

Glutamic acid 95.69 ± 1.52a 70.84 ± 3.70b 40.00 ± 4.08c

Glycine 95.06 ± 2.05a 66.55 ± 6.26b 16.82 ± 5.30c

Histidine 94.33 ± 1.69a 74.26 ± 2.86b 20.70 ± 6.02c

Isoleucine 93.23 ± 1.72a 68.72 ± 3.99b 37.95 ± 5.92c

Leucine 92.23 ± 1.96a 71.29 ± 3.16b 35.17 ± 5.32c

Lysine 95.03 ± 1.84a 76.97 ± 2.24b 40.13 ± 3.88c

Methionine 95.20 ± 1.54a 70.63 ± 3.30b 48.31 ± 3.06c

Phenylalanine 93.41 ± 1.90a 65.28 ± 4.13b 34.43 ± 5.37c

Proline 94.68 ± 1.92a 67.21 ± 5.39b 4.86 ± 7.02c

Serine 93.11 ± 1.91a 58.31 ± 4.65b 20.24 ± 5.47c

Threonine 91.99 ± 1.94a 66.33 ± 3.35b 40.16 ± 3.84c

Tryptophan 95.37 ± 1.92a 80.31 ± 1.53b -34.14 ± 24.88c

Tyrosine 95.28 ± 1.22a 73.62 ± 3.40b 44.04 ± 7.25c

Valine 90.78 ± 2.39a 67.06 ± 3.75b 49.27 ± 6.00c

Total 94.31 ± 1.67a 69.91 ± 3.89b 34.87 ± 4.71c

Values from each treatment are means and standard deviation of triplicate tanks. Values within a row different superscripts are significantly different (P < 0.05).

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CHAPTER VI

EVALUATION OF A FISH MEAL ANALOGUE AS A REPLACEMENT FOR FISH

MEAL IN PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei

1. Introduction

Fish meal (FM) is the most important and preferred protein source in most shrimp feeds,

because it is an excellent source of essential nutrients such as protein and indispensable amino

acids, essential fatty acids, cholesterol, vitamins, minerals, attractants and unidentified growth

factors (Samocha et al., 2004; Swick et al., 1995). Because of FM’s comparatively high

nutritional value and limitation of availability, it has a high demand and limit supply resulting in

a corresponding high market value. Hence, we must shift our emphasis and use this ingredient

only when nutrient requirements of the animal demand its use (Davis and Sookying, 2009).

As commercial shrimp culture has become an expanded and intensified economic activity,

the demand for cost effective protein sources continues to increase because there is a

considerable cost saving in shifting protein sources when it is economical and nutritionally

viable. Partial replacement of FM in the diets for Pacific white shrimp were demonstrated to be

feasible in many studies (Cruz-Suárez et al., 2007; Goytortúa-Bores et al., 2006; Ju et al., 2012;

Liu et al., 2012; Oujifard et al., 2012; Tan et al., 2005). If the substitution strategy considers

shifts in essential nutrients, it also appears that FM can be removed from shrimp formulations if

suitable alternative sources of protein and lipids are provided to meet the nutrient requirements

of the animal (Davis et al., 2004). The use of complementary ingredients is a practice used to

obtain a more balanced nutrient profile in the feeds (i.e. essential amino acids, fatty acids) and to

increase nutrient utilization and facilitate feed processing (Samocha et al., 2004). Based on

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numerous studies with Pacific white shrimp reared under a variety of culture conditions and

densities, FM can be successfully removed from shrimp feed formulations in properly balanced

production diets without compromising the growth of shrimp (Amaya et al., 2007a;b; Browdy et

al., 2006; Roy et al., 2009; Samocha et al., 2004; Sookying and Davis, 2011).

Aqua-Pak Pro-Cision is a blend of animal and plant protein products supplemented with

encapsulated essential amino acids and some other essential nutrients that designed for shrimp

industry as a substitution for FM. Because it has a similar balanced nutrient profile in terms of

protein, lipid, and amino acids to that of FM and has a lower price it may be suitable for

inclusion in the shrimp diets. The information of this product as a FM replacement in practical

diets for the Pacific white shrimp is still limited. Hence, the objectives of this study were to

determine the nutrient digestibility values of the ingredient and evaluate the potential effects of

this product as a replacement for FM in practical diets for Pacific white shrimp, L. vannamei.

2. Materials and Methods

2.1. Experimental design and diets

All test diets were formulated to be isonitrogenous and isolipidic (35% protein and 8%

lipid). In the growth trial 1 and 2, five experimental diets were formulated to contain increasing

level of FMA (0, 4.85, 9.70, 14.55, and 19.44%) as a replacement of FM (0, 5, 10, 15, and 20%)

(Table 1). In trial 3, five experimental diets were essentially the same as the diets used in trial 1

and 2, but balanced for P (Table 2). Additionally, a reference diet (Table 3) was utilized to

determine digestibility coefficients in conjunction with 1% chromic oxide as an inert marker and

70:30 replacement strategy.

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Primary ingredients were analyzed at University of Missouri Agricultural Experiment

Station Chemical Laboratories (Columbia, MO, USA) for proximate composition, P content, and

amino acid profile (Table 4). All experimental diets were produced at the Aquatic Animal

Nutrition Laboratory at the School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn

University (Auburn, AL, USA) using the standard procedures for the shrimp feeds. Briefly, diets

were prepared by mixing the pre-ground dry ingredients in a food mixer (Hobart, Troy, OH,

USA) for 10–15 minutes. Hot water was then blended into the mixture to obtain a consistency

appropriate for pelleting. Diets were pressure-pelleted using a meat grinder with a 2.5-mm die.

The wet pellets were then placed into a fan-ventilated oven (< 50 °C) overnight in order to attain

a moisture content of less than 10%. Dry pellets were crumbled, packed in sealed bags, and

stored in a freezer until use. The diets were analyzed at University of Missouri Agricultural

Experiment Station Chemical Laboratories (Columbia, MO, USA) and Midwest Laboratories

(Omaha, NE, USA) for proximate composition, amino acid profile, and mineral composition

(Table 5 and 6).

2.2. Growth trials

Three trials were conducted at the E.W. Shell Fisheries Research Station, Auburn

University (Auburn, AL, USA). Pacific white shrimp post larvae (PL) were obtained from

Shrimp Improvement Systems (Islamorada, Florida) and nursed in an indoor recirculating

system. PLs were fed a commercial feed (Zeigler Bros., Inc., Gardners, Pennsylvania, USA)

using an automatic feeder for ~1 week, and then switched to crumbled commercial shrimp feed

(Zeigler Bros., Inc., Gardners, Pennsylvania, USA) for ~1- 2 weeks.

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In trial 1 and 2, the recirculating system consisted of 30 aquaria (135 L) connected to a

common reservoir, biological filter, bead filter, fluidized biological filter and recirculation pump.

Six replicate groups of shrimp (0.47 g initial mean weight, 10 shrimp/tank for trial 1; 0.40 g

initial mean weight, 10 shrimp/tank for trial 2) were offered diets using our standard feeding

protocol over 6 weeks. Based on historic results, feed inputs were pre-programmed assuming the

shrimp would double their weight weekly up to one gram then gain 0.8-1.1 g weekly with a feed

conversion ratio (FCR) of 1.8. Daily allowances of feed were adjusted based on observed feed

consumption, weekly counts of the shrimp and mortality. Consequently, for each tank in trial 1, a

fixed ration of 1.21 g day-1 for the first week, 2.06 g day-1 for the second week, 2.31 g day-1 for

the third week, 2.57 g day-1 for the fourth week, and 2.83 g day-1 for the remaining culturing

period was offered, partitioned in 4 feedings each day. For each tank in trial 2, a fixed ration of

1.03 g day-1 for the first week, 2.06 g day-1 for the second to fourth week, 2.31 g day-1 for the

fifth week, and 2.57 g day-1 for the last week was also allotted in 4 portions per day.

In trial 3, the recirculating system consisted of 20 aquaria (80 L) connected to a common

reservoir, biological filter, bead filter, fluidized biological filter and recirculation pump. Four

replicate groups of shrimp (0.25 g initial mean weight, 10 shrimp/tank) were offered diets using

our standard feeding protocol over 6 weeks. Based on historic results, feed inputs were pre-

programmed assuming the shrimp would double their weight weekly up to one gram then gain

0.8-1.1 g weekly with a feed conversion ratio (FCR) of 1.8. Daily allowances of feed were

adjusted based on observed feed consumption, weekly counts of the shrimp and mortality.

Consequently, for each tank in trial 3, a fixed ration of 0.68 g day-1 for the first week, 1.36 g day-

1 for the second week, 2.18 g day-1 for the third and fourth week, and 2.73 g day-1 for the

remaining culturing period was offered, partitioned in 4 feedings each day.

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At the conclusion of each growth trial, shrimp were counted and group-weighted. Mean

final weight, FCR, WG, biomass, and survival were determined (Table 7). After obtaining the

final total weight of shrimps in each aquarium, 4 shrimps from trial 2 and trial 3 were randomly

selected and frozen at -20 °C for subsequent determination of whole body composition.

Proximate and mineral compositions of whole shrimp body in trial 2 and trial 3 (Table 9 and

Table 10) were analyzed by University of Missouri-Columbia, Agriculture Experiment Station

Chemical Laboratory (Columbia, MO, USA) and Midwest Laboratories (Omaha, NE, USA),

respectively. Protein and mineral retention was calculated as follows:

Protein retention (%) = (final weight × final protein content) - (initial weight × initial protein

content) × 100 / protein offered.

Mineral retention (%) = (final weight × final mineral content) - (initial weight × initial mineral

content) × 100 / mineral offered.

2.3. Water quality monitoring

For all trials, dissolved oxygen (DO), water temperature and salinity were measured

twice daily by using a YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA).

Hydrogen potential (pH) was measured twice weekly by using a waterproof pHTestr30 (Oakton

instrument, Vernon Hills, IL, USA). Total ammonia-nitrogen (TAN) and nitrite were evaluated

every week by using the methods described by Sororzano (1969) and Spotte (1979).

2.4. Digestibility trial

The digestibility trial was conducted in the mentioned recirculation system and utilized

six shrimp per aquaria with six aquaria per dietary treatment. Once acclimated for three days to

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the test diets, feces from two aquaria were pooled (n=3) and collected over a 5-day period or

until adequate samples were obtained. To obtain fecal samples, the aquaria were cleaned by

siphoning before each feeding with the first collection of the day discarded. After cleaning, the

shrimp were offered an excess of feed and then about 1 hour later feed was removed and feces

were collected by siphoning onto a 500µm mesh screen. Collected feces were rinsed with

distilled water, dried at 105 °C until a constant weight was obtained, and then stored in freezer

(−20 °C) until analyzed. Apparent digestibility coefficient for dry matter, protein, energy, and

amino acids were determined by using chromic oxide (Cr2O3, 10 g kg -1) as an inert marker.

Chromium concentrations were determined by the method of McGinnis and Kasting (1964) in

which, after a colorimetric reaction, absorbance is read on a spectrophotometer (Spectronic

genesis 5, Milton Roy Co., Rochester, NY, USA) at 540nm. Gross energy of diets and fecal

samples were analyzed with a Semi micro-bomb calorimeter (Model 1425, Parr Instrument Co.,

Moline, IL, USA). Protein were determined by micro-Kjeldahl analysis (Ma and Zuazaga, 1942).

Amino acids were analyzed by University of Missouri-Columbia, Agriculture Experiment

Station Chemical Laboratory (Columbia, MO, USA). The apparent digestibility coefficient of

dry matter (ADMD), protein (APD), energy (AED), and amino acids (AAAD) were calculated

according to Cho et al. (1982) as follows:

ADMD (%) = 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces)]

APD, AED, and AAAD (%) =100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces) × (% nutrient in

feces / % nutrient in feed]

The apparent digestibility coefficients (ADC) of the test ingredients for dry matter,

energy, protein and amino acids were calculated according to Bureau, Hua (2006) as follows:

ADCtest ingredient = ADCtest diet + [(ADCtest diet – ADCref. diet) × (0.7 × Dref / 0.3 × Dingr)]

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where Dref = % nutrient (or KJ/g gross energy) of reference diet mash (as is); Dingr = % nutrient

(or KJ/g gross energy) of test ingredient (as is).

2.5. Statistical analysis

All the data were analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). Data from

were analyzed using one-way ANOVA to determine significant differences (P<0.05) among

treatments followed by the Tukey’s multiple comparison test to determine difference between

treatments in each trial. Pearson correlation analysis was performed to determine if there is a

correlation between dietary P levels and biological responses of shrimp. The pooled standard

errors were used across growth trials, as the variance of each treatment is the same.

3. Results

3.1. Water quality

In trial 1, DO, temperature, salinity, pH, TAN, and nitrite were maintained at

6.04±0.42mg L-1, 27.9±0.4°C, 9.7±0.3ppt, 7.8±0.2, 0.083±0.088mg L-1, and 0.020±0.019mg L-1,

respectively. In trial 2, DO, temperature, salinity, pH, TAN, and nitrite were maintained at

5.86±0.31mg L-1, 28.5±0.8°C, 8.2±1.4ppt, 7.5±0.3, 0.041±0.050mg L-1, and 0.058±0.102mg L-1,

respectively. In trial 3, DO, temperature, salinity, pH, TAN, and nitrite were maintained at

6.66±0.3 mg L-1, 28.8 ± 2.4 °C, 8.0 ± 0.9 ppt, 7.0 ± 0.3, 0.029 ± 0.036 mg L-1, and 0.121 ± 0.128

mg L-1, respectively. Water quality conditions in all trials were suitable for normal growth and

survival of this species.

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3.2. Growth trial

Growth performance of juvenile Pacific white shrimp fed with experimental diets are

presented in Table 7. In trial 1, shrimp offered diets incorporated with 4.85% FMA replacement

exhibited significantly higher WG than treatment contained 14.55% FMA. No significantly

differences were observed in final biomass (34.68g to 37.10g), final mean weight (4.29 g to

4.85g), FCR (2.21 to 2.55), and survival (75% to 86.7%). In trial 2, shrimp fed with diets

contained 4.85 and 9.7% FMA showed significantly higher final biomass, final mean weight, and

WG, but lower FCR than shrimp offered diets supplemented with 19.4% FMA. No significant

difference was found in survival (81.7% to 91.7%). In trial 3, final mean weight and WG were

significantly improved in the treatment contained 4.85% FMA compared to the treatment

supplemented with 14.55% FMA. No significant differences were determined in biomass (35.28

to 42.35 g), FCR (1.68 to 2.20), and survival (80.0 to 87.5%).

Pearson correlation coefficients of growth performance, FCR, and survival with dietary P

levels are presented in Table 8. In trial 1 and trial 2, dietary phosphorus levels had significant

positive correlation with weight gain while negatively correlated with FCR. No correlations of

phosphorus levels with survival were observed.

Proximate and mineral compositions of whole shrimp body as well as protein and P

retention in trial 2 and trial 3 are presented in Table 9 and Table 10. In trial 2, shrimp fed with

diet containing 4.85% FMA exhibited significantly higher lipid content compared to treatment

included with 19.4% FMA. Protein content was significantly improved when shrimp was fed

with diet contained 19.4% FMA in contrast with treatments contained 0 and 4.85% FMA.

Shrimp fed with diets containing 4.85 and 9.7% FMA exhibited significantly improved protein

retention (PR) compared to those fed with the diet supplemented with 19.4% FMA. No

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significant differences were detected in moisture (72.28 to 75.97%), crude fiber (5.71 to 6.25%),

ash (11.85 to 12.45%), and P (1.03 to 1.08%) contents of whole shrimp body and P retention

(11.49 to 14.09%) across the treatments.

In trial 3, significantly improved PR was detected in the shrimp fed diets containing

4.85% FMA. P retention in the treatment supplemented with 19.4% FMA was significantly

higher than that in the treatment containing 14.55% FMA. No significant differences were

determined in protein (74.23 to 75.8%), moisture (76.81 to 78.26%), lipid (4.65 to 5.82%), ash

(11.40 to 12.07%), sulfur (0.84 to 0.9%), phosphorus (0.99 to 1.06%), potassium (1.33 to

1.46%), magnesium (0.26 to 0.3%), calcium (3 to 3.61%), sodium (1.07 to 1.19%), iron (17.1 to

26.03 mg kg-1), manganese (2.68 to 3.53 mg kg-1), copper (69.4 to 75.85 mg kg-1), zinc (74 to

77.2 mg kg-1) contents in the whole shrimp body.

3.3. Digestibility trial

Apparent dry matter (ADM), apparent energy (AED), and apparent protein (APD)

digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered

to shrimp are presented in Table 11. The digestibility trial contained a range of ingredients;

hence, we have provided a few other ingredients as a reference. In terms of the proximate

composition, FMA contains a higher protein and lipid content, but lower phosphorus level than

FM (Table 2). In order to confirm the results, fecal samples for basal diets and FM diet were

recollected. The results turned out to be quite similar, which indicated that the feces collection

and samples analysis methods we utilized in the digestibility study are consistent. The energy

and protein digestibility of FMA were 53.5% and 32.4%, respectively, which were significantly

lower than those of FM.

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Apparent amino acids (AA) digestibility values for the SBM, FM, and FMA using 70:30

replacement technique offered to Pacific white shrimp are presented in Table 12. Most individual

amino acids compositions in FMA are higher than those in FM. In terms of two of the most

limiting AAs in shrimp feeds, FMA shared similar methionine and lysine levels as FM (4.99% vs

4.67% and 1.69% vs 1.61%, respectively). The AAs digestibility corresponded to the protein

digestibility. Apparent digestibility coefficients of alanine, arginine, aspartic acid, cysteine,

glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,

serine, threonine, tryptophan, tyrosine, and valine were of FMA were significantly lower than

those of FM and SBM.

4. Discussion

The nutrient composition of FMA compared to FM exhibited higher crude protein

(68.28% vs 62.78%) and lipid (13.89% vs 10.56%) contents, but lower P level (1.68% vs

3.15%). Generally, both essential and non-essential amino acids concentrations in FMA are

superior to FM. When FM is replaced with alternative protein sources, it is crucial to balance

essential nutrients especially amino acids in the diets. Proximate compositions, amino acids

profiles, and mineral contents (excluding P levels) are consistent in all the treatments of the

growth trials (Table 5 and 6). There is a decreasing trend of P level as the inclusion level of FMA

increases in the diets for trial 1 and trial 2 (Table 5) due to the comparatively lower P content in

this ingredient. All diets in trial 3 shared a similar P content as P level was balanced by adding

inorganic P in formulation (Table 6).

The nutrient digestibility of a feed ingredient is an important factor to evaluate the overall

nutritive value of the ingredient because it is related to the quantity of the nutrient absorbed by

144

the animals. In the current study, APD and AED of FMA were significantly lower than those of

SBM and FM. The comparatively lower APD of FMA translated to lower AAAD. In general,

there was a reasonable correspondence of the AAAD to APD of FMA. SBM had the highest

APD (97.03%), AED (82.56%), and ADAA (90.78 – 96.91%) among the ingredients tested in

the current study. Similar ranges of results for APD, AED, and AAAD were reported in multiple

shrimp studies (Cruz-Suarez et al., 2009; Fang et al., 2016; Liu et al., 2013; Yang et al., 2009;

Zhou et al., 2015). ADP and ADE of FM1 were 67.07% and 69.77%, respectively. Similar

results were acquired in FM2 (ADP and ADE: 71.3% and 65.78%, respectively). The analogous

results of basal diet and FM diet from the collection of two fecal samples pointed to the

consistency in the feces collection and sample analysis methods. APD of FM has been reported

to be ranged from 62.7% to 91.6% in numerous studies (Lemos et al., 2009; Liu et al., 2013;

Terrazas-Fierro et al. 2010; Yang et al., 2009). ADP of FM in the present study were in the

lower range of the results documented among those researches. The differences could be

attributed to several factors, such as different raw materials, location or processing methods used

to produce the products, and unknown factors related to different production batches.

With regards to the growth trials, significantly reduced growth was observed in the diet

contained 14.55% FMA compared to the one supplemented with 4.85% FMA. If the growth

depression resulted from the FMA inclusion levels, the diet contained highest inclusion of FMA

should also be negatively affected. However, no difference in terms of growth was detected in

the treatment contained 19.4% FMA. Given the relatively lower survival in trial 1, the growth

results may be masked. Hence, trial 2 was initiated by applying the same diets to confirm the

results. At the end of trial 2, shrimp fed with diet contained 19.4% FMA exhibited significantly

lower WG and higher FCR than those fed with diets supplemented with 4.85 and 9.7% FMA.

145

There is a clear decreasing trend when inclusion level of FMA increased to 14.55% of the diet.

Given the balanced proximate composition and amino acid profile across the treatments, the

reduced P level might be attributed to depressed growth at the high inclusion levels of FMA.

Correlation analysis outputs (Table 8) also indicated that P level in the diets positively correlated

with WG and negatively correlated with FCR in both trial 1 and trial 2, which demonstrated our

nutritional assumption from statistical standpoint.

P is an important constituent of nucleic acids and cell membranes, a major constituent of

structural components of structural tissues, and is directly involved in all energy-producing

cellular reactions (NRC, 2011). Dietary P deficiency impairs intermediary metabolism, which

results in reduced growth and increased feed conversion. P requirement of Pacific white shrimp

is affected both by P and calcium (Ca) levels as well as presence of inhibitors (Cheng et al.,

2006; Davis et al., 1993). Cheng et al. (2006) indicated diets contained 0.93% total P in the

absence of Ca were adequate for optimal growth of Pacific white shrimp. In the current study, P

levels in diets supplemented with 14.55% and 19.4% FMA are 0.82% and 0.78%, respectively,

which may suggest P limitation in these two diets. To elucidate if decreased P was the cause of

the growth depression in trial 1 and trial 2, the third trial was conducted to essentially utilize the

same diets in trial 1 and 2, but balanced for P. Results indicated that shrimp fed with 4.95%

FMA performed significantly better in terms of growth than those fed with diets contained

14.55% FMA. No clear decreasing trend of growth was detected when diets contained FMA

were compared to the reference diet (20% FM based diet). At a conclusion of the growth trials, P

levels in the diets may limit the growth when shrimp fed with diets contained 14.55% and 19.4%

FMA in trial 1 and 2. However, reasons for the improvements in growth when FMA was

supplemented at 4.95% in all trials are not able to be defined.

146

There are numerous studies looking at the potential of alternative ingredients as FM

replacement in shrimp feeds. The results varied as different ingredient and reference diets

utilized. In general, many researches documented that partial FM replacement by alternative

ingredients in shrimp feeds was applicable (Cruz-Suárez et al., 2007; Goytortúa-Bores et al.,

2006; Ju et al., 2012; Liu et al., 2012; Oujifard et al., 2012; Suárez et al., 2009; Tan et al., 2005;

Yue et al., 2012). The failure of complete FM replacement can be attributed to many factors such

as palatability problem, imbalanced essential nutrients (protein, lipid, essential amino acids, P,

and etc.), and relatively lower nutrient availability than FM. Hence, choosing suitable alternative

ingredients and considering shifts in essential nutrients in the substitution strategy would be the

key factors to the success of complete FM replacement. In proper balanced shrimp diets,

complete FM can be replaced by suitable alternative ingredients which were reported by multiple

researchers (Amaya et al., 2007a;b; Bauer et al., 2012; Browdy et al., 2006; Roy et al., 2009;

Samocha et al., 2004; Sookying and Davis, 2011).

In the present study, lipid content of whole shrimp body was significantly decreased in

the diet contained 19.4% FMA compared to the one supplemented with 4.95% FMA in trial 2,

which may due to the comparatively lower energy availability in FMA than FM. Whereas the

protein content of whole shrimp body was dramatically improved in the diet supplemented with

19.4% FMA in contrast with the ones incorporated with 0 and 4.95% FMA, which may be a

result of reduced lipid content of shrimp as no significant difference were observed in moisture,

crude fiber, and ash content of shrimp body. Similarly, Hernández et al. (2008) indicated that

protein content of shrimp body was significantly enhanced while lipid level was significantly

reduced when porcine meat meal was incorporated in the diet to replace FM. On the contrary, no

significant differences were detected in the protein and lipid contents of shrimp body in trial 3

147

when shrimp were fed with the essentially same diets except for different P levels as in trial 2.

As energy digestibility of FMA was not dramatically lower than FM, its effect on the lipid

content may be marginal. Accordingly, no significant differences were detected in protein and

lipid contents of shrimp body in FM replacement studies (Oujifard et al., 2012; Ju et al., 2012;

Yue et al., 2012).

Although there is a decreasing trend of P content in the diets, no significant difference

was observed in the P level in shrimp body in trial 2. Similarly, Ju et al. (2012) documented that

P level of shrimp body was not affected when there was a decreasing trend of P content in the

diets using a defatted microalgae meal to replace FM. Hepatopancreas and carapace are two

good indicator tissues of the mineral status of shrimp (Davis et al., 1993). As the concentrations

of these two tissues were diluted in the whole body samples, the reflection of P status in shrimp

might be masked. In trial 3, no significant difference was detected in the P content of shrimp

body as the diets were balanced for P level.

Nutrient retention was determined by a number of factors including dietary nutrient

levels, feed intake, final weight and initial weight of animals as well as the final and initial

nutrient content of animals (Halver and Hardy, 2002). In trial 2, shrimp fed diets containing 4.85

and 9.7% FMA exhibited improved PR in contrast with those offered the diet containing 19.4%

FMA. No differences were detected in dietary protein levels, feed offered to the shrimp, and

initial weight of shrimp. Although protein content of whole shrimp body was significantly

improved in the diet contained 19.4% FMA, its effect cannot counteract with the significantly

reduced final weight of shrimp in the same treatment, which would be the primary cause of

reduced PR. In trial 3, results demonstrated that PR in the treatment supplemented with 4.95%

FMA was significantly improved, resulting by the significantly enhanced final mean weight.

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Similarly, Hernández et al. (2008) reported PR was significantly reduced when shrimp fed with

diets supplemented with high levels of porcine meat meal to replace FM, resulting from the

significantly depressed final weight even though protein content of shrimp body was enhanced.

In both trial 2 and trial 3, no significant differences were detected in the P retention.

5. Conclusion

Under the conditions of this work, FMA can replace FM up to 20% in practical shrimp

diets supplemented with inorganic P without compromising the growth of shrimp. P limitation is

likely the reason for growth depression in the treatment devoid of FM when diets were not

balanced for P. The improvement of growth when FMA was incorporated at 4.95% across three

trials was not able to be defined. Given the good growth across the range of inclusion without

any indication of a growth depression, the digestibility of the protein of FMA would be similar to

that of the FM for which it was substituted. The low nutrient digestibility of FMA may due to an

atypical response or the product simply does not work with the testing technique.

149

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154

Table 1 Composition (% as is) of test diets used in the growth trial 1 and 2.

Ingredient Diet code

D1 D2 D3 D4 D5

Fish meal1 20.00 15.00 10.00 5.00 0.00

Soybean meal2 37.20 37.20 37.20 37.20 37.20

Whole wheat3 32.00 32.00 32.00 32.00 32.00

Fish meal analogue4 0.00 4.85 9.70 14.55 19.40

Fish oil2 4.39 4.26 4.13 4.00 3.87

Trace mineral premix5 0.50 0.50 0.50 0.50 0.50

Vitamin premix6 1.80 1.80 1.80 1.80 1.80

Choline chloride3 0.20 0.20 0.20 0.20 0.20

Stay C7 0.10 0.10 0.10 0.10 0.10

Lecithin8 1.00 1.00 1.00 1.00 1.00

Corn starch3 2.81 3.09 3.37 3.65 3.93 1 Menhaden fish meal, special select. Omega Protein Inc., Houston, TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 MP Biomedicals Inc., Solon, OH, USA. 4 Aqua-Pak Pro-Cision, H. J, Baker Brother Inc., Shelton, CT, USA. 5 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 Enhanced D97, The Solae Company, St. Louis, MO, USA.

155

Table 2 Composition (% as is) of test diets used in the growth trial 3.

Ingredient Diet code

D1p D2p D3p D4p D5p

Fish meal1 20.00 15.00 10.00 5.00 0.00

Soybean meal2 37.30 37.30 37.30 37.30 37.30

Whole wheat3 34.00 34.00 34.00 34.00 34.00

Fish meal analogue4 0.00 4.85 9.70 14.55 19.40

Fish oil2 4.54 4.37 4.19 4.02 3.87

Trace mineral premix5 0.50 0.50 0.50 0.50 0.50

Vitamin premix6 1.80 1.80 1.80 1.80 1.80

Choline chloride3 0.20 0.20 0.20 0.20 0.20

Stay C7 0.10 0.10 0.10 0.10 0.10

Lecithin8 1.00 1.00 1.00 1.00 1.00

Cholesterol3 0.05 0.05 0.05 0.05 0.05

Mono-dicalcium phosphate9 0.00 0.50 0.75 1.20 1.50

Corn starch3 0.51 0.33 0.41 0.28 0.48 1 Menhaden fish meal, special select. Omega Protein Inc., Houston, TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 MP Biomedicals Inc., Solon, OH, USA. 4 Aqua-Pak Pro-Cision, H. J, Baker Brother Inc., Shelton, CT, USA. 5 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 Enhanced D97. The Solae Company, St. Louis, MO, USA. 9 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA.

156

Table 3 Composition of reference diet for the determination of digestibility coefficients of fish meal analogue (FMA), fish meal (FM), and soybean meal (SBM). Ingredients % as is

Soybean meal1 10.0

Fish meal2 32.5

Fish oil2 3.2

Whole wheat3 47.6

Trace mineral premix4 0.5

Vitamin premix5 1.8

Choline cloride6 0.2

Stay C7 0.1

Corn starch3 1.0

Lecethin8 1.0

Chromic oxide9 1.0 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Menhaden fish meal, special select. Omega Protein Inc., Houston TX, USA. 3 MP Biomedicals Inc., Solon, Ohio, USA 4 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 5 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 6 VWR, Radnor, PA, USA. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 Enhanced D97. The Solae Company, St. Louis, MO, USA. 9 Alfa Aesar, Haverhill, MA, USA.

157

Table 4 Proximate composition1, phosphorus content1, and amino acid profile1 of the ingredients used in the growth and digestibility trials.

Composition (% as is) Fish meal analogue Fish meal Soybean meal Crude protein 68.28 62.78 44.89 Moisture 4.95 7.99 10.97 Crude fat 13.89 10.56 3.78 Crude fiber 0.80 0.00 3.20 Ash 12.54 18.75 6.67 Phosphorus 1.68 3.15 0.66 Alanine 4.38 3.91 2.04 Arginine 4.21 3.68 3.35 Aspartic acid 5.22 5.34 5.1 Cysteine 1.4 0.47 0.62 Glutamic acid 7.46 7.47 8.24 Glycine 5.15 4.88 2.04 Histidine 1.9 1.63 1.2 Isoleucine 2.7 2.42 2.17 Leucine 5.47 4.21 3.57 Lysine 4.99 4.67 3.06 Methionine 1.69 1.61 0.66 Phenylalanine 3.19 2.39 2.35 Proline 4.17 3.08 2.39 Serine 3.12 2.11 1.9 Taurine 0.22 0.73 0.13 Threonine 2.87 2.41 1.75 Tryptophan 0.39 0.62 0.62 Tyrosine 2.04 1.67 1.64 Valine 4.11 2.99 2.34 1 Ingredients were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA).

158

Table 5 Proximate composition1 (% as is), mineral composition (% as is: phosphorus, sulfur, potassium, magnesium, calcium, sodium; mg kg-1 as is: iron, manganese, copper, zinc)2 and amino acid profile1 (% as is) of the test diets used in the growth trial 1 and 2.

Composition D1 D2 D3 D4 D5 Crude protein 36.82 36.66 36.70 36.36 36.90 Moisture 6.76 7.36 7.37 7.61 7.49 Crude fat 7.62 7.66 7.31 7.14 8.00 Crude fiber 4.57 3.96 4.51 3.89 4.05 Ash 7.42 6.78 6.38 5.93 5.48 Phosphorus 1.10 1.01 0.92 0.84 0.78 Sulfur 0.43 0.42 0.43 0.43 0.46 Potassium 1.29 1.19 1.15 1.11 1.12 Magnesium 0.22 0.21 0.21 0.20 0.20 Calcium 1.33 1.32 1.11 0.93 0.80 Sodium 0.20 0.17 0.15 0.13 0.10 Iron 270 278 299 314 336 Manganese 57.7 57.0 55.6 55.0 56.3 Copper 16.4 18.7 15.4 15.8 16.5 Zinc 227 210 204 212 209 Alanine 1.74 1.71 1.74 1.80 1.80 Arginine 2.2 2.15 2.22 2.27 2.29 Aspartic Acid 3.22 3.11 3.16 3.21 3.21 Cysteine 0.42 0.45 0.51 0.57 0.61 Glutamic Acid 6.27 6.13 6.23 6.23 6.23 Glycine 1.98 1.94 1.98 1.93 1.91 Histidine 0.89 0.88 0.90 0.94 0.98 Isoleucine 1.60 1.56 1.59 1.66 1.65 Leucine 2.61 2.6 2.64 2.84 2.86 Lysine 2.21 2.17 2.22 2.25 2.33 Methionine 0.63 0.65 0.66 0.69 0.70 Phenylalanine 1.64 1.66 1.71 1.81 1.82 Proline 2.13 2.15 2.22 2.29 2.28 Serine 1.13 1.18 1.19 1.26 1.29 Taurine 0.24 0.22 0.20 0.17 0.15 Threonine 1.22 1.21 1.22 1.28 1.3 Tryptophan 0.40 0.39 0.38 0.34 0.34

159

Tyrosine 1.08 1.09 1.13 1.17 1.18 Valine 1.78 1.78 1.84 1.99 2.00 1 Proximate composition and amino acid profiles of test diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA). 2 Mineral composition was tested at Midwest Laboratories (Omaha, NE, USA).

160

Table 6 Proximate composition1 (% as is), mineral composition (% as is: phosphorus, sulfur, potassium, magnesium, calcium, sodium; mg kg-1 as is: iron, manganese, copper, zinc)2 and amino acid profile1 (% as is) of the test diets used in the growth trial 3. Composition1 D1p D2p D3p D4p D5p Moisture 6.92 6.73 7.61 7.58 7.90 Protein 36.60 37.40 36.80 36.20 36.20 Fat 7.86 8.06 7.70 7.41 7.42 Fiber 5.10 6.70 5.60 5.90 6.50 Ash 7.34 7.59 7.28 7.25 6.88 Sulfur 0.41 0.43 0.43 0.4 0.38 Phosphorus 1.09 1.29 1.17 1.12 1.03 Potassium 1.17 1.19 1.15 1.05 0.97 Magnesium 0.21 0.22 0.22 0.19 0.18 Calcium 1.44 1.82 1.55 1.44 1.27 Sodium 0.19 0.19 0.15 0.12 0.09 Iron 214 276 269 285 288 Manganese 65.1 62.1 61.8 67.6 57.6 Copper 13.1 16.1 14.1 25.3 12.7 Zinc 270 188 177 173 144 Alanine 1.78 1.77 1.83 1.77 1.77 Arginine 2.25 2.22 2.24 2.22 2.21 Aspartic Acid 3.26 3.23 3.27 3.19 3.18 Cysteine 0.44 0.46 0.53 0.52 0.55 Glutamic Acid 6.43 6.30 6.22 6.18 6.08 Glycine 2.01 2.05 2.06 2.02 2.00 Histidine 0.88 0.90 0.98 0.95 0.98 Isoleucine 1.50 1.48 1.43 1.41 1.36 Leucine 2.59 2.60 2.75 2.73 2.81 Lysine 2.33 2.38 2.41 2.36 2.37 Methionine 0.67 0.70 0.70 0.67 0.68 Phenylalanine 1.62 1.63 1.69 1.68 1.72 Proline 2.02 2.06 2.13 2.17 2.14 Serine 1.49 1.43 1.53 1.56 1.65 Threonine 1.33 1.29 1.31 1.30 1.31 Tryptophan 0.46 0.45 0.44 0.42 0.41

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Tyrosine 1.12 1.09 1.09 1.07 1.06 Valine 1.74 1.80 1.93 1.96 1.99 1 Amino acid profiles of test diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA). 2 Proximate composition and mineral content were tested at Midwest Laboratories (Omaha, NE, USA).

162

Table 7 Performance of juvenile Pacific white shrimp L. vannamei (Initial weight: 0.47, 0.40, 0.25 g in trial 1, 2, and 3, respectively) offered diets with different fish meal analogue (FMA) levels (0, 4.85, 9.70, 14.55, and 19.44%) replacing different levels of fish meal for six weeks.

Trial Diet FMA

levels (%)

Biomass

(g)

Final mean

weight (g) WG (%)2 FCR3 Survival

(%)

Trial

1

n=6

D1 0 35.62 4.56 881ab 2.35 78.3

D2 4.85 36.35 4.85 978a 2.21 75.0

D3 9.70 37.10 4.32 854ab 2.51 86.7

D4 14.55 35.63 4.29 774b 2.55 83.3

D5 19.40 34.68 4.37 839ab 2.47 80.0

PSE1 1.8373 0.1545 15.6790 0.0953 4.6904

P value 0.9116 0.0915 0.0163 0.1046 0.467

Trial

2

n=6

D1 0 37.10ab 4.36a 973ab 2.22b 85.0

D2 4.85 38.20ab 4.60a 1027a 2.10b 83.3

D3 9.70 41.55a 4.53a 1013a 2.10b 91.7

D4 14.55 33.88bc 3.92 ab 883ab 2.54ab 86.7

D5 19.40 29.70c 3.64b 812b 2.73a 81.7

PSE1 0.8008 0.0730 19.6751 0.0498 1.2397

P value 0.0032 0.0030 0.0184 0.0027 0.2063

Trial

3

n=4

D1p 0 37.70 4.38ab 1689.0ab 2.11 87.5

D2p 4.85 42.35 5.32a 2039.4a 1.68 80.0

D3p 9.70 38.80 4.73ab 1848.4ab 1.99 82.5

D4p 14.55 36.25 4.13b 1515.5b 2.20 87.5

D5p 19.40 35.28 4.43ab 1634.8ab 2.08 80.0

PSE1 1.2521 0.1228 55.5090 0.0621 3.0448

P value 0.3511 0.0350 0.0382 0.0779 0.8142 1 PSE: Pooled standard error. 2 WG: Weight gain = (Final weight-initial weight)/initial weight*100.

163

3 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

164

Table 8 Pearson correlation coefficients of final biomass, final mean weight, weight gain (WG), feed conversion ratio (FCR), and survival in trial 1 and trial 2 with phosphorus levels of the diets. The first line of each cell is the value of correlation coefficient and the second line of each cell is P-value.

Trial Final

biomass

Final mean

weight WG FCR Survival

Trial 1 Phosphorus 0.0758 0.3393 0.3840 -0.3481 -0.1627

0.6905 0.0666 0.0362 0.0594 0.3904

Trial 2 Phosphorus 0.4220 0.5251 0.4662 -0.5335 0.0343

0.0202 0.0029 0.0094 0.0024 0.8571

165

Table 9 Whole body proximate composition (% dry weight basis), phosphorus content (P, % dry weight basis), and protein and phosphorus retention (%) when shrimp were offered diets supplemented with different fish meal analogue levels replacing fish meal without balanced for phosphorus for six weeks in trial 2.

Diet Protein Moisture Lipid Fiber Ash P PR2 PHR3

D1 72.84b 75.83 8.22ab 6.25 12.45 1.07 23.58ab 11.49

D2 72.99b 75.28 8.65a 5.84 11.97 1.08 25.80a 13.84

D3 73.76ab 75.97 7.72ab 6.09 12.14 1.03 25.49a 14.09

D4 73.99ab 75.94 7.64ab 5.71 11.85 1.06 21.88ab 13.43

D5 74.53a 76.79 7.09b 5.73 12.17 1.03 18.91b 12.17

P-value 0.0051 0.0909 0.0348 0.4326 0.8888 0.2026 0.0029 0.2921

PSE1 0.1314 0.1460 0.1383 0.0981 0.1759 0.0073 0.4995 0.0689 1 PSE: Pool standard error. 2 PR: Protein retention = (final weight × final protein content) - (initial weight × initial protein content) × 100 / protein offered. 3 PHR: Phosphorus retention = (final weight × final phosphorus content) - (initial weight × initial phosphorus content) × 100 / phosphorus offered. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

166

Table 10 Whole body proximate1 composition, mineral composition1, and protein and phosphorus retention when shrimp were offered diets supplemented with different fish meal analogue levels replacing different levels of fish meal balance for phosphorus for six weeks in trial 3.

Diet D1 D2 D3 D4 D5 PSE2 P-value

Proximate composition (% dry weight basis)

Protein 74.57 75.80 75.13 75.53 74.23 0.5048 0.8028

Moisture 77.68 76.81 76.98 78.26 77.33 0.5145 0.8629

Lipid 5.32 5.82 5.80 4.65 5.78 0.2273 0.3272

Ash 12.07 11.95 11.45 11.93 11.40 0.1652 0.5337

Macro elements (% dry weight basis)

Sulfur 0.90 0.89 0.87 0.85 0.84 0.0072 0.0751

Phosphorus 1.01 1.03 0.99 1.00 1.06 0.0111 0.2544

Potassium 1.46 1.45 1.37 1.40 1.33 0.0161 0.0725

Magnesium 0.3 0.29 0.27 0.27 0.26 0.0052 0.2387

Calcium 3.61 3.57 3.18 3.36 3.00 0.1026 0.2641

Sodium 1.19 1.10 1.08 1.11 1.07 0.0161 0.2048

Trace elements (mg kg-1 dry weight basis)

Iron 21.70 17.10 18.40 26.03 24.6 1.3880 0.1636

Manganese 3.53 2.68 3.10 2.73 3.10 0.2136 0.6902

Copper 70.63 69.40 69.88 69.63 75.85 1.2789 0.3866

Zinc 77.2 75.53 74.00 74.40 74.83 0.7098 0.6344

Retention (%)

Protein3 22.95b 28.38a 23.78b 20.92b 22.91b 0.4442 0.0006

Phosphorus4 10.37 11.03 9.77 8.81 11.40 0.2670 0.2288 1 Body samples were analyzed at tested at Midwest Laboratories (Omaha, NE, USA). 2 PSE: Pool standard error. 3 Protein retention = (final weight × final protein content) - (initial weight × initial protein content) × 100 / protein offered. 4 Phosphorus retention = (final weight × final phosphorus content) - (initial weight × initial phosphorus content) × 100 / phosphorus offered. Values within a row with different superscripts are significantly different based on Tukey’s multiple range test.

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van

nam

ei.

A

DM

D

AED

D

APD

D

AD

MD

I A

EDI

APD

I

Bas

al1

76.3

8 ±

0.37

a 82

.66

± 1.

20a

92.0

8 ±

0.55

a

B

asal

2 75

.69

±0.5

2a 81

.51

± 0.

41a

92.0

4 ±

0.03

a

SBM

77

.02

± 0.

87a

82.6

3 ±

1.05

a 94

.76

± 0.

49a

78.5

1 ±

2.89

a 82

.56

± 3.

79a

99.0

0 ±

1.27

a

FM1

68.2

1 ±

3.80

b 78

.31

± 3.

21ab

80

.86

± 1.

80b

49.1

5 ±

12.6

7b 69

.77

± 9.

51a

67.0

7 ±

4.02

b

FM2

67.9

9 ±

0.17

b 76

.44

± 0.

78bc

82

.34

± 0.

31b

49.4

5 ±

0.56

b 65

.78

± 2.

23ab

71

.30

± 0.

68b

FMA

66

.06

± 1.

67b

72.3

4 ±

1.67

c 64

.00

± 2.

14c

41.9

8 ±

5.58

b 53

.53

± 4.

71b

32.3

9 ±

4.55

c

1 PS

E: P

ool s

tand

ard

erro

r. V

alue

s fr

om e

ach

treat

men

t ar

e m

eans

and

sta

ndar

d de

viat

ion

of t

riplic

ate

tank

s. V

alue

s w

ithin

a c

olum

n di

ffer

ent

supe

rscr

ipts

are

si

gnifi

cant

ly d

iffer

ent (

P <

0.05

).

168

Table 12 Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal (FM), poultry meal (PM), and fish meal analogue (FMA) using 70:30 replacement technique offered to Pacific white shrimp L. vannamei.

AA digestibility

coefficients (%) SBM FM FMA

Alanine 93.75 ± 2.02a 69.09 ± 4.09b 39.23 ± 2.89c

Arginine 96.91 ± 1.44a 75.35 ± 3.78b 42.66 ± 2.71c

Aspartic acid 95.39 ± 1.36a 69.23 ± 3.70b 34.38 ± 3.57c

Cysteine 91.29 ± 1.68a 54.39 ± 7.06b 23.73 ± 5.72c

Glutamic acid 95.69 ± 1.52a 70.84 ± 3.70b 43.48 ± 3.86c

Glycine 95.06 ± 2.05a 66.55 ± 6.26b 50.14 ± 2.93c

Histidine 94.33 ± 1.69a 74.26 ± 2.86b 32.72 ± 3.35c

Isoleucine 93.23 ± 1.72a 68.72 ± 3.99b 31.55 ± 3.41c

Leucine 92.23 ± 1.96a 71.29 ± 3.16b 30.58 ± 3.24c

Lysine 95.03 ± 1.84a 76.97 ± 2.24b 54.13 ± 2.40c

Methionine 95.20 ± 1.54a 70.63 ± 3.30b 72.49 ± 2.01c

Phenylalanine 93.41 ± 1.90a 65.28 ± 4.13b 29.28 ± 3.66c

Proline 94.68 ± 1.92a 67.21 ± 5.39b 38.77 ± 3.13c

Serine 93.11 ± 1.91a 58.31 ± 4.65b 37.23 ± 2.99c

Threonine 91.99 ± 1.94a 66.33 ± 3.35b 34.41 ± 3.37c

Tryptophan 95.37 ± 1.92a 80.31 ± 1.53b 61.88 ± 6.20c

Tyrosine 95.28 ± 1.22a 73.62 ± 3.40b 48.02 ± 3.60c

Valine 90.78 ± 2.39a 67.06 ± 3.75b 26.77 ± 2.23c

Total 94.31 ± 1.67a 69.91 ± 3.89b 40.42 ± 3.14c

1 Pooled standard error. Values from each treatment are means and standard deviation of triplicate tanks. Values within a row different superscripts are significantly different (P < 0.05).

169

CHAPTER VII

UTILIZATION OF GREEN SEAWEED ULVA sp. AS A PROTEIN SOURCE IN

PRACTICAL DIETS FOR PACIFIC WHITE SHRIMP Litopenaeus vannamei

1. Introduction

Traditionally, fish meal (FM) is the most important and preferred protein source in most

shrimp feeds, because it is an excellent source of essential nutrients such as protein and

indispensable amino acids, essential fatty acids, cholesterol, vitamins, minerals, attractants and

unidentified growth factors (Samocha et al., 2004). As most fisheries are beyond sustainable

limits, fish meal supplies will not increase. Hence, the high demand and limit supply resulted in a

corresponding high market value of FM. The price of FM has risen from around $400 per metric

ton in 2000 to more than $1700 per metric ton in 2016, and the highest price reached to $2388

per metric ton in 2014 (Index Mundi, 2016).

Shrimp nutritionists have investigated numerous FM alternatives to reduce the reliance

on this costly and potential limiting resources in shrimp feed formulation. Among the potential

alternative ingredients, soybean meal (SBM) is usually considered as the most reliable, cost-

effective, and nutritionally valuable protein source in shrimp feed. The popularity of soybean

meal as a protein source is the result of a well-balanced nutrient profile, high digestibility, stead

supply, expandable production and reasonable price (Davis and Arnold, 2000, Amaya et al.,

2007a, Amaya et al., 2007b). The successful application of SBM in practical feed formulation

for Pacific white shrimp were well documented in many studies (Amaya et al., 2007a, Amaya et

al., 2007b, Davis and Arnold, 2000, Sookying and Davis, 2011, Roy et al., 2009, Samocha et al.,

2004, Qiu and Davis, 2016b, Qiu and Davis, 2016a).

170

Although FM and SBM will continue to be a mainstay in shrimp formulation in terms of

their nutritional preponderances, it is also critical to explore alternative ingredients to these two

conventional protein sources that may be more economical and sustainable to support the rapid

expansion of the shrimp industry as shrimp consumption is expected to continue to increase.

Marine macro-algae, commonly referred to seaweeds, are categorized by their pigmentation,

morphology, anatomy, and nutritional composition as red, brown or green seaweeds

(Dawczynski et al., 2007). They can benefit from recycling waste carbon dioxide (CO2) from

combustive energy production and waste nutrients produced by intensive aquaculture operations,

intensive animal operations, and municipal waste treatment (Kaushik, 2010, Sargent and Tacon,

1999). In an integrated cultivation system, the macro-algae use the metabolic residues of animals

as nutrients, absorb CO2 and produce O2 for the environment (Marinho-Soriano et al., 2007). The

interaction allows the excretion of an organism to serve as food for another. Presently, significant

improvements in growth and survival rate have been observed when Pacific white shrimp,

Litopenaeus vannamei (Cruz-Suárez et al., 2010, Brito et al., 2014a, Brito et al., 2014b), giant

tiger shrimp, Penaeous monodon Fabr (Tsutsui et al., 2010, Izzati, 2012), and yellowleg shrimp,

Farfantepenaeus californiensis (Portillo�Clark et al., 2012) are co-cultured with seaweeds.

Ulva spp belongs to the green seaweed. A number of studies have demonstrated that

dietary Ulva meal inclusion at low levels (<5% of the diet) did not affect the growth performance

in a variety of species including African catfish, Clarias gariepinus (Abdel-Warith et al., 2016),

gilthead seabream Sparus aurata (Emre et al., 2013), Pacific white shrimp (Cárdenas et al.,

2015, Rodríguez-González et al., 2014), Nile tilapia Oreochromis niloticus (Güroy et al., 2007,

Ergün et al., 2009), rainbow trout Oncorhynchus mykiss (Güroy et al., 2013). However, the

171

growth response of different fish species to the moderate and high supplementation levels of

Ulva meal are somewhat inconsistent.

The information about green seaweed Ulva sp. as a protein source in shrimp feed

formulation is still limited. Therefore, the purposes of this study were to evaluate the potential of

Ulva sp. as a protein source in comparison to FM and SBM in practical diets for Pacific white

shrimp and investigate the nutrient availability in this ingredient.

2. Materials and Methods

2.1. Experimental diets

Primary ingredients and four pooled batches of sun dried Ulva pertussa meal were

analyzed for proximate composition, amino acids and minerals (Table 1 and 2) and the diets

were formulated. Additionally, prior to pooling batch two, the 7 individual daily collections of

Ulva meal were sampled and analyzed individually (Table 3). Upon completion of analysis diets

were made by weighing pre-grounding dry ingredients and oil which was then mixed in a food

mixer (Hobart Corporation, Troy, OH, USA) for 15 min. Hot water was then blended into the

mixture to obtain a consistency appropriate for pelleting. Diets were pressure-pelleted using a

meat grinder with a 3-mm die, air-dried (< 45 °C) to a moisture content of 8-10%. Pellets were

crumbled, packed in sealed plastic bags and stored in a freezer until needed.

In trial 1, 2, 3 and 4, diets were formulated to be isonitrogenous and isolipidic (35%

protein and 8% lipid). In most diets substitution was done on a protein to protein basis however

in trial 3, ingredient replacement was done on a digestible protein basis. In trial 1, five

experimental diets were formulated to contain increasing levels (0, 6.35, 12.70, 19.05, and

25.40%) of the first batch of Ulva meal (UM1) as a replacement for fish meal (Table 4a). In trial

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2, nine experimental diets were formulated (Table 5a). The basal diet for this and subsequent

trials was designed to have 6% fishmeal in all formulations to help stabilize nutrients as well as

palatability. The first seven diets utilized increasing levels of the second batch of Ulva meal

(UM2) (0, 5, 10, 15, 20, 25, and 30%) to replace soybean meal. Diet 8 and Diet 9 utilized high

incorporation of Ulva meal from the first and third batch, respectively. This allowed a

comparison of all three meals at equivalent levels of protein replacement. In trial 3, four

experimental diets were formulated using the first three batches of Ulva meal replacing soybean

meal on a digestible protein basis (Table 6a). In trial 4, five experimental diets were designed

utilizing different levels (0, 4.75, 9.5, 12, and 24%) of fourth batch of Ulva meal (high protein

content~38%) replacing fish meal and soybean meal. Digestibility values for the meals were

determined using standard methods and 1% chromic oxide as an inert marker (Table 8a). Test

diets were made on a dry matter basis using a 70:30 mixture of the reference diet and test

ingredients.

The ingredients (Table 1) were analyzed at University of Missouri Agricultural

Experiment Station Chemical Laboratories (Columbia, MO, USA) for proximate composition

and amino acid profile and mineral profiles (Table 2) by the Soil Lab (Auburn, AL, USA). Daily

collections of UM2 samples (Table 3) collected across seven different dates were analyzed

individually at Midwest laboratories (Omaha, NE, USA) for proximate and mineral composition.

Diets (Table 4a, Table 5a, Table 6a, and Table 7a) were analyzed at University of Missouri

Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA) for proximate

composition and amino acid profile and Soil Lab (Auburn, AL, USA) for mineral composition.

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2.2. Growth trials

The trial 1 utilized 5 treatments with 7 replicates in each treatment. It was conducted in a

semi-closed recirculation system. Juvenile shrimp were obtained from the nursery system and

selected by hand-sorting to a uniform size. Juvenile shrimp (initial weight 0.26 ± 0.02 g) were

stocked into 35 tanks with 10 shrimp in each aquarium (135L). A sub-sample of shrimp from the

initial stocking was retained for whole body analysis to be utilized for later protein retention

analysis. As shrimp are difficult to handle, intermittent weights were not taken. However, shrimp

were counted to readjust daily feed input on a weekly basis. Based on historically results, a fixed

ration was calculated assuming a 1.8 feed conversion ratio and a doubling in size the first two

weeks and 0.8-0.9 g week-1. Therefore, for ten shrimp in a given tank, a fixed ration of 0.67 g

day-1 for the first week, 1.45 g day-1 for the second week, 2.06 g day-1 for the third week, and

2.31 g day-1 for the fourth week, 2.57 g day-1 for the fifth week, and 2.83 g day-1 for the six week

was offered over 4 feedings.

The trial 2 was conducted in the same semi-closed recirculation system which is

mentioned above. It utilized 9 treatments with 4 replicates in each treatment. Juvenile shrimp

(initial weight 0.24 ± 0.01 g) were stocked into 36 tanks with 10 shrimp in each aquarium

(135L). A sub-sample of shrimp from the initial stocking was retained for whole body samples to

be utilized for later protein and phosphorus retention analysis. Shrimp were counted to readjust

daily feed input on a weekly basis. Based on historically results, a fixed ration was calculated

assuming a 1.8 feed conversion ratio and a doubling in size the first two weeks and 0.8-0.9 g

week-1 thereafter. Consequently, for each tank a fixed ration of 0.62 g day-1 for the first week,

1.23 g day-1 for the second week, 2.06 g day-1 for the third and fourth week, and 2.31 g day-1 for

the fifth week was offered over 4 feedings.

174

The trial 3 was conducted in the same semi-closed recirculation system which is

mentioned above. It utilized 4 treatments with 4 replicates in each treatment. Juvenile shrimp

(initial weight 0.98 ± 0.01 g) were stocked into 16 tanks with 10 shrimp in each aquarium

(135L). A sub-sample of shrimp from the initial stocking was retained for whole body samples to

be utilized for later protein and phosphorus retention analysis. As shrimp are difficult to handle,

intermittent weights were not taken. However, shrimp were counted to readjust daily feed input

on a weekly basis. Based on historically results, a fixed ration was calculated assuming a 1.8

feed conversion ratio and a doubling in size the first two weeks and 0.8-1.3 g week-1 thereafter.

Consequently, for each tank of 10 shrimp a fixed ration of 2.1 g day-1 for the first week, 2.3 g

day-1 for the second week, 2.8 g day-1 for the third week, 3.1 g day-1 for the fourth week, 3.4 g

day-1 for the fifth week, and 3.7 g day-1 for the sixth week was offered over 4 feedings.

The trial 4 was conducted in a similar semi-closed recirculation system to what was

previously described. It utilized 5 treatments with 4 replicates in each treatment. Juvenile shrimp

(initial weight 0.15 ± 0.01 g) were stocked into 20 tanks with 10 shrimp in each aquarium

(135L). Based on historically results, a fixed ration was calculated assuming a 1.8 feed

conversion ratio and a doubling in size the first two weeks and 0.8-1.3 g week-1 thereafter.

Consequently, for each tank of 10 shrimp a fixed ration of 2.1 g day-1 for the first week, 2.3 g

day-1 for the second week, 2.8 g day-1 for the third week, 3.1 g day-1 for the fourth week, 3.4 g

day-1 for the fifth week, and 3.7 g day-1 for the sixth week was offered over 4 feedings.

At the end of each growth trials, shrimp were counted and group weighted. Mean final

weight, feed conversion ratio (FCR), weight gain (WG), biomass, and survival were determined

(Table 4c, 5d, 6c, 7c). After obtaining the final total weight of shrimps in each aquarium, 4

shrimps were randomly selected and frozen at -20 °C for subsequent determination of whole

175

body composition. Proximate composition (Table 4d, 5e, 6d, 7d) of whole shrimp was analyzed

by Midwest Laboratories, Inc (Omaha, NE, USA) or University of Missouri Agricultural

Experiment Station Chemical Laboratories (Columbia, MO, USA). Mineral profiles of whole

shrimp and select algae meals were analyzed by Soils lab (Auburn, AL, USA). Apparent net

protein and amino acids retention were calculated using the following equations

Protein retention (%) = (final weight × final protein content) - (initial weight × initial protein

content) × 100 / protein offered.

Amino acids (AA) retention (%) = (final weight × final AA content) - (initial weight × initial AA

content) × 100 / AA offered.

2.3. Water quality monitoring

Dissolved oxygen (DO), temperature, and salinity were measured twice daily by using a

YSI 650 multi-parameter instrument (YSI, Yellow Springs, OH, USA). The pH was measured

twice weekly by using a waterproof pHTestr30 (Oakton instrument, Vernon Hills, IL, USA).

Water samples were taken to measure total ammonia-nitrogen (TAN) and nitrite every week.

TAN and nitrite were determined by the methods described by (Solorzano, 1969) and (Spotte,

1979), respectively.

2.4. Digestibility trial

The digestibility trial was conducted in the mentioned recirculation system and utilized 6

shrimp per aquaria with 6 aquaria per dietary treatment. Once acclimated for 3 days to the test

diets, feces from two aquaria were pooled (n=3) and collected over a 5-day period or until

adequate samples were obtained. To obtain fecal samples, the aquaria were cleaned by siphoning

176

before each feeding with the first collection of the day discarded. After the aquaria were cleaned,

the shrimp were offered an excess of feed and then about 1 hour later feed was removed and

feces were collected by siphoning onto a 500µm mesh screen. Collected feces were rinsed with

distilled water, dried at 105 °C until a constant weight was obtained, and then stored in freezer (-

20 °C) until analyzed. Apparent digestibility coefficient for dry matter, protein, energy and

amino acids were determined by using chromic oxide (Cr2O3, 10 g kg -1) as an inert marker.

Chromium concentrations were determined by the method of (McGinnis and Kasting, 1964) in

which, after a colorimetric reaction, absorbance is read on a spectrophotometer (Spectronic

genesis 5, Milton Roy Co., Rochester, NY, USA) at 540nm. Gross energy of diets and fecal

samples were analyzed with a Semi micro-bomb calorimeter (Model 1425, Parr Instrument Co.,

Moline, IL, USA). Protein were determined by micro-Kjeldahl analysis (Ma and Zuazaga, 1942).

The apparent digestibility coefficient of dry matter (ADMD), protein (ADP), energy (ADE) and

amino acids (ADAA) were calculated according to (Cho et al., 1982)as follows:

ADMD (%) = 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces)]

ADP, ADE, and ADAA (%) = 100 − [100 × (% Cr2O3 in feed / % Cr2O3 in feces) × (% nutrient

in feces / % nutrient in feed).

The apparent digestibility coefficients (ADC) of the test ingredients for dry matter,

energy, protein and amino acids were calculated according to Bureau et al. (2006) as follows:

ADCtest ingredient = ADCtest diet + [(ADCtest diet – ADCref. diet) × (0.7 × Dref / 0.3 × Dingr)]

where Dref = % nutrient (or KJ/g gross energy) of reference diet mash (as is); Dingr = % nutrient

(or KJ/g gross energy) of test ingredient (as is).

177

2.5. Statistical analysis

All the data were analyzed using SAS (V9.3. SAS Institute, Cary, NC, USA). Data from

both the growth trial and digestibility trial were analyzed using one-way ANOVA to determine

significant differences (P<0.05) among treatments followed by the Tukey’s multiple comparison

test to determine difference between treatments. Arcsine square root transformation was used

prior to analysis for the proportion data. False discover rate (FDR) controlling procedures were

applied to adjust the P-value to control the FDR for amino acid data. Linear, second- or third-

order polynomial regressions were performed to investigate the relationship between the

supplemental Ulva meal levels and weight gain, FCR, survival, and lipid content of whole

shrimp body. To identify the most appropriate regression model, we compared P-value of the

model components, R2 value, adjust R2 value, and the sum of squares for error (SSE) with

different regression models.

3. Results

3.1. Water quality

In trial 1, DO, temperature, salinity, pH, TAN, and nitrite were maintained within

acceptable ranges for L. vannamei at 6.19 ± 0.25 mg L-1, 28.4 ± 0.8 °C, 11.8 ± 0.4 ppt, 7.23 ±

0.22, 0.079 ± 0.041 mg L-1, and 0.039 ± 0.021 mg L-1, respectively. In trial 2, DO, temperature,

salinity, pH, TAN, and nitrite were maintained at 5.82 ± 0.26 mg L-1, 29.7 ± 0.8 °C, 8.6 ± 0.4

ppt, 7.5 ± 0.5, 0.052 ± 0.107 mg L-1, and 0.003 ± 0.004 mg L-1, respectively. In trial 3, DO,

temperature, salinity, pH, TAN, and nitrite were maintained at 6.20 ± 0.72 mg L-1, 29.5 ± 0.9 °C,

8.4 ± 1.0 ppt, 7.5 ± 0.3, 0.092 ± 0.103 mg L-1, and 0.050 ± 0.039 mg L-1, respectively. In trial 4,

DO, temperature, salinity, pH, TAN, and nitrite were maintained at 6.96 ± 0.31 mg L-1, 28.1 ±

178

0.3 °C, 8.2 ± 0.6 ppt, 7.0 ± 0.3, 0.05 ± 0.04 mg L-1, and 0.12 ± 0.12 mg L-1, respectively. Water

quality conditions in all the trials were suitable for normal growth and survival of this species.

3.2. Growth performances

Growth performances of juvenile Pacific white shrimp offered diets contained different

levels of Ulva meal in trial 1-4 are presented in Table 4c, 5d, 6c, and 7c. In trial 1, final biomass

was significantly reduced when UM1 was included at 25.4% compared to the treatment

supplemented with 0 and 6.35% UM1. Significant reductions in final mean weight and

increments in FCR were detected when 19.05 and 25.4% UM1 was incorporated in the diet in

contrast with diets contained 0 and 6.35% UM1. Shrimp fed with diets contained more than

12.7% UM1 exhibited significantly reduced WG than those offered with diets supplemented with

0 and 6.35% UM1. No significant difference was determined in survival across all the treatments

(82.9% to 92.9%).

In trial 2, final biomass was significantly reduced when more than 5% Ulva meal was

included in the diet. Final mean weight and WG were significantly decreased when more than

10% Ulva meal was supplemented in the diet. Significant increment in FCR was determined

when the diet contained 25% UM2. Shrimp fed with diets contained 25% and 30% UM2

exhibited significantly lower survival than those fed with diets supplemented with 0, 5, and 15%

UM2.

In trial 3, in general shrimp fed UM2 exhibited poorest performance in terms of growth,

FCR, and survival. Significant reductions in final biomass, final mean weight, WG, and survival

were determined when UM2 was supplemented in the diet compared to other treatments. FCR

was significantly increased in the treatment contained UM2 in contrast with other treatments.

179

In trial 4, shrimp fed with diets supplemented with different levels of UM4 showed

significantly reduced final biomass, final mean weight, and WG as well as increased FCR.

Survival was significantly reduced when 9.5% UM4 was included in the diet to replace 6% FM.

3.3. Proximate composition and amino acid profile of whole shrimp body

Proximate composition and amino acids profile of whole shrimp body in trial 1-4 are

presented in Table 4d, 5e, 6d, and 7d. In trial 1, shrimp fed with diets supplemented with

different levels of UM1 exhibited significantly reduced crude lipid of whole body. No significant

effects were detected in moisture (76.29% to 76.99%) and crude protein content (72.77% to

74.27%) across the treatments.

In trial 2, crude lipid content was significantly reduced when more than 5% Ulva meal

was incorporated in the diets. Shrimp fed with diets contained more than 5% UM2 exhibited

significantly improved crude protein content. No significant difference was detected in the

moisture content (75.66% to 78.05%) across all the treatments.

Shrimp fed with diet contained 25% and 30% exhibited significantly higher arginine and

glycine content than the one fed with reference diet. Cysteine and lysine contents in the treatment

fed with 15% to 30% UM2 were significantly higher than those fed with the reference diet.

Histidine content in the treatment fed with diet contained 10% to 25% UM2 and 23.6% UM3

was significantly higher than the ones fed with reference diet. Shrimp fed with diet contained

30% UM2 exhibited significantly higher methionine content than those fed with diets contained

0, 5, and 10% UM2. Phenylalanine in the treatment contained 20% UM2 was significantly

higher than the treatments contained 0 and 5% UM2 as well as 26.3% UM1. No significant

differences were observed in alanine, aspartic acid, glutamic acid, hydroxylysine,

180

hydroxyproline, isoleucine, leucine, proline, serine, threonine, tyrosine, tryptophan, valine, and

total amino acids levels in whole shrimp body across all the treatments.

In trial 3, significant reduction in crude lipid content was detected when shrimp was fed

with diet contained UM2. Shrimp fed with diet contained UM2 exhibited significantly higher

body moisture content than those fed with reference diet and diet contained UM4. No significant

difference was detected in protein content (75.08% to 76.98%) across all the treatments.

Methionine content was significantly improved when UM1, UM2, UM3 were included in

the diets. No significant differences were observed in alanine, arginine, aspartic acid, cysteine,

glutamic acid, glycine, histidine, hydroxylysine, hydroxyproline, isoleucine, leucine, lysine,

phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and total amino acids

contents of whole shrimp body across all the treatments.

In trial 4, moisture content in the treatment fed with diets contained 12% and 24% UM4

was significantly higher than those fed with reference diet and diet contained 4.75% UM4. Crude

protein was significantly enhanced when shrimp were fed with diet contained 24% UM4

compared to the reference diet and diet supplemented with 4.75% UM4. Crude lipid was

dramatically decreased when more than 9.5% UM4 was included in the diet. Significant

increment in ash content was observed when the diet was incorporated with 9.5% and 24% UM4

in contrast with the reference diet.

3.4. Protein and amino acids retention

Protein and amino acids retention in trial 1-4 are presented in Table 4c, 5f, 6e, and 7c. In

trial 1, shrimp fed diet contained 6.35% UM1 showed significantly improved PRE compared to

181

those fed with diets supplemented with 19.05 and 25.4% UM1. In trial 4, PRE was significantly

depressed when more than 9.5% UM4 was include in the diet

In trial 2, PRE was significantly reduced when shrimp was fed with diets supplemented

with 15 to 30% UM2, 26.3% UM1, and 23.6%UM3 compared to the one offered with reference

diet. In general, total and individual amino acids retention corresponded to PRE. Alanine,

arginine, aspartic acid, hydroxyproline, isoleucine, phenylalanine, proline, threonine, and valine

retention were significantly lower in the treatments contained 15 to 30% UM2, 26.3% UM1, and

23.6%UM3 than the reference diet. Total amino acids, cysteine, glutamic acid, glycine, leucine,

and serine retention were significantly reduced in the treatments contained 15 to 30% UM2 and

23.6% UM3. Shrimp fed with diets contained 15 to 30% UM2 exhibited significantly higher

histidine, hydroxylysine, lysine, methionine, tryptophan, and tyrosine retention than those fed

with reference diet.

In trial 3, PRE was significantly reduced when UM2 and UM3 were supplemented in the

diets. In general, total and individual amino acids retention corresponded to PRE. Arginine and

hydroxyproline retention were significantly lower in treatments incorporated with UM1-3. Total

amino acids, cysteine, serine, threonine, and valine retention in treatments supplemented with

UM2 and UM3 were significantly reduced compared to the treatments fed with reference diet.

Alanine, aspartic acid, glycine, histidine, hydroxylysine, isoleucine, leucine, lysine, methionine,

phenylalanine, proline, tryptophan, and tyrosine retention were significantly depressed when

UM2 was supplemented in the diet.

182

3.5. Regression analysis

Dietary Ulva meal levels significantly correlated with WG, FCR, and lipid content in trial

1, 2, and 4 (Figure 1, 2, and 3). In general, the trends of the relationships between

WG/FCR/lipid content of shrimp body and dietary Ulva meal levels are consistent in trial 1, 2,

and 4. WG is negatively correlated with dietary Ulva meal levels. In trial 1, 2, and 4, the

regression lines are described by y = 0.1686x3 - 6.2114x2 + 34.409x + 1797.8 (R2 = 0.4922, P <

0.0001), y = 1.1925x2 -62.699x + 1713.1 (R2 = 0.6815, P < 0.0001), and y = 2.6848x2 - 119.17x

+ 3049.3 (R2 = 0.6934, P < 0.0001), respectively (y = WG, x = Ulva meal levels). FCR is

positively associated with Ulva meal levels. In trial 1, 2, and 4, the regression lines are exhibited

as y = 0.0276x + 1.7708 (R2 = 0.3582, P = 0.0001), y = -0.0703x + 1.5451 (R2 = 0.4766, P <

0.0001), and y = 0.06x + 1.8532 (R2 = 0.721, P < 0.0001), respectively (y = FCR, x = Ulva meal

levels). Lipid content of shrimp body is negatively correlated with Ulva meal levels. In trial 1, 2,

and 4, the regression lines are described by y = -0.0962x + 7.4 (R2 = 0.3503, P = 0.0002), y =

0.0078x2 - 0.4095x + 7.8033 (R2 = 0.9051, P < 0.0001), and y = -0.1903x + 7.9555 (R2 = 0.7224,

P < 0.0001), respectively. Survival is negatively correlated with dietary Ulva meal levels in trial

2 and 4. However, no relationship is determined in trial 1. In trial 2 and 4, the regression lines are

described by y = -1.1607x + 100.27 (R2 = 0.6113, P < 0.0001) and y = -0.7625x + 90.105 (R2 =

0.2479, P = 0.0156).

3.6. Digestibility trial

Apparent dry matter (ADM), apparent energy (AED), and apparent protein (APD)

digestibility values for the diet (D) and ingredient (I) using 70:30 replacement technique offered

to shrimp are presented in Table 8b. The digestibility trial contained a range of ingredients;

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hence, we have provided a few other ingredients as a reference. In order to confirm the results,

fecal samples for basal diets and FM diet were recollected. The results turned out to be quite

similar, which indicated that the feces collection and samples analysis methods we utilized in the

digestibility study are consistent. The energy and protein digestibility of UM1 and UM2 were

40.39% and 15.17%, and 19.11% and 43.51%, respectively, which were significantly lower than

those of FM and SBM.

Apparent amino acids (AA) digestibility values for the SBM, FM, UM1, and UM2 using

70:30 replacement technique offered to Pacific white shrimp are presented in Table 8c. In

general, the AAAD corresponded to the APD. Apparent digestibility coefficients of alanine,

arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine,

methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine of UM1

and UM2 were significantly lower than those of FM and SBM.

4. Discussion

4.1. Ingredient composition

Seaweeds are valuable sources of protein, fiber, vitamins, macro and trace elements, as

well as important bioactive compounds (Ortiz et al., 2006). Protein content of seaweeds is tied to

the variations in seasons (Fleurence, 1999). In the current study, differences in protein contents

were detected among the four pooled batches of Ulva meal (Table 1) and individual Ulva meal

samples collected from seven dates within the UM2 (Table 2). The protein content of seaweed

can be somewhat manipulated by controlling the nutrient loading during cultivation (Floreto et

al., 1996). In this instance, nitrogen enriched conditions like the effluents of fish or shrimp

farms, where seaweeds are utilized as bio-filters, can enhance their protein contents (Lahaye et

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al., 1995, Pinchetti et al., 1998). Owing to the manipulation of nitrogen loading during

cultivation, the protein content of seaweed Ulva sp. can be improved up to 38.16% in the present

study, which is superior to the upper limit of the protein range (10% to 26%) of this species

reported by several researchers (Benjama and Masniyom, 2011, Fleurence, 1999, Lee et al.,

2014).

As a result of the enhanced protein content, the amino acid concentrations in the Ulva sp.

reported in the present study were improved at least by around 50% in contrast with those

documented by Lee et al. (2014) or by even more than 300% compared to those described by

Benjama and Masniyom (2011). For most seaweed species, aspartic acid and glutamic acid

constitute a large portion of the total amino acids (Lourenço et al., 2002). Similarly, the sum of

these two amino acids in UM1 to UM4 represented 22.27% to 28.6% of total amino acids in the

present study. In terms of the two most limiting amino acids (methionine and lysine) in

ingredients for shrimp, their concentrations in UM1-4 ranged from 0.26% to 0.63% and 0.82% to

1.51%, respectively. Methionine level in UM1-4 were similar to that in SBM (0.66%) but lower

than that in FM (1.61%). Lysine level in UM1-4 were low compared to both SBM (3.06%) and

FM (4.67%).

Lipid content in most seaweeds was reported less than 4% dry weight (DW), whereas

some seaweeds such as Dictyota acutiloba and D. sandvicenis contained a high level of lipid

(16.1% and 20.2 % DW, respectively) (McDermid and Stuercke, 2003). In the current study,

lipid content in UM1-4 and individual UM2 samples ranged from 0 to 0.62%. Similarly, Lee et

al. (2014) reported that Lipid content of Ulva sp. was 0.1% DW. By contrast, Benjama and

Masniyom (2011) documented that lipid content of Ulva sp. in summer and rainy season were

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7.4% and 2.1% DW, respectively. The differences among these researches would be mainly

attributed to different nutrient loadings during the seaweed cultivation.

Seaweeds are rich sources of minerals with a broad mineral composition including Al,

As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S, Se, Si, and Zn, which has not

been observed in edible terrestrial plants (Lee et al., 2014). In the present study, the macro

elements (Ca, K, Mg, Na, P, and S) and trace elements (Al, As, B, Ba, cd, Co, Cr, Cu, Fe, Mn,

Ni, Pb, Se, Si, Zn, and Zr) of pooled UM1-3 ranged from 0.31-4.79% and 0.6-9086.7 mg kg-1,

respectively. Within the individual UM2 samples collected from seven dates, the macro elements

(Ca, K, Mg, Na, P, and S) and trace elements (Cu, Fe, Mn, and Zn) ranged from 0.31-4.38% and

7.6-6780 mg kg-1, respectively. Considerable variations were detected in the Ca, Na, and Fe

levels in the pooled UM1-3 and individual UM2 samples, which would be mainly attributed to

the nutrient loadings, temperature, and salinity during cultivation.

4.2. Nutrient digestibility

The nutrient digestibility of a feed ingredient is an important factor to evaluate the overall

nutritive value of the ingredient because it is related to the quantity of the nutrient absorbed by

the animals. SBM had the highest APD (97.03%), AED (82.56%), and AAAD (90.78 – 96.91%)

among the ingredients tested in the current study. Similar ranges of results for APD, AED, and

AAAD were reported in multiple shrimp studies (Cruz-Suárez et al., 2009, Fang et al., 2016, Liu

et al., 2013, Yang et al., 2009, Zhou et al., 2015). APD and AED of FM1 were 67.07% and

69.77%, respectively. Similar results were acquired in FM2 (APD and AED: 71.3% and 65.78%,

respectively). The analogous results of basal diet and FM diet from the collections under two

occasions pointed to the consistency in the feces collection and sample analysis methods. APD

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of FM has been reported to be ranged from 62.7% to 91.6% in numerous studies (Lemos et al.,

2009, Liu et al., 2013, Terrazas-Fierro et al., 2010, Yang et al., 2009). ADP of FM in our study

were in the lower range of the results documented among those researches. The differences in

digestibility of FM among various studies could be attributed to several factors, such as different

raw materials, location or processing methods used to produce the products, and unknown

factors related to different production batches.

In the present study, APD and AED of UM1 and UM2 were significantly lower than

those of FM and SBM. The low APD of UM1 and UM2 translated to poor AAAD. Total amino

acids and most individual amino acids availability in UM1 and UM2 were significantly lower

than those of FM and SBM. With regards to the two batches of Ulva meal, UM1 exhibited

significantly higher AED than that of UM2. On the contrary, APD of UM1 was significantly

lower than that of UM2. Because of comparatively low APD in UM1, ADC of total amino acids

and many individual amino acids in UM1 were significantly lower than those in UM2. No

differences were detected in ADMD of UM1 and UM2. The variations in APD, AED, and

AAAD of UM1 and UM2 indicated that nutrient availability in Ulva meal would also be affected

by the conditions such as temperature, salinity, nutrient loadings during cultivation.

There are relatively few studies looking at the nutrient availability of seaweed meals in

aquatic animal feeds particularly with regards to shrimp. Cárdenas et al. (2015) documented that

APD of Nutrikelp (a brown seaweed meal is comprised of mixtures of Macrocystis,

Lessoniaceae and Lessonia) and Nutrigreen (a green seaweed meal contains mixtures of Ulva,

Caulerpa, and Enteromorpha) for L. vannamei were 85.37% and 86.81%, respectively, which

may be masked by their high ADMD (80.97% and 80.33%, respectively) accordingly. Moreover,

Pereira et al. (2012) reported that AED, and APD of four seaweeds (Ulva spp, Porphyra dioica,

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Gracilaria vermiculophylla, and Sargassum muticum) in rainbow trout Oncorhynchus mykiss

were 72.7% and 75.6%, 66.8% and 79.5%, 62.4% and 87.8%, and 58% and 65.5%, respectively.

In the same study, the AED and APD of the four seaweeds in Nile tilapia Oreochromis niloticus

were 57.1% and 63.4%, 39.6% and 58.5%, 27.8% and 51.4%, and 54.9% and 65.1%,

respectively (Pereira et al., 2012). The variations in the nutrients availability results presented in

the researches could be mainly attributed to the use of multiple seaweed species and different

aquatic animals in the experiment.

There are several other studies investigated the nutrient availability of the test diets

supplemented with seaweed meals. Valente et al. (2006) indicated that supplementation of three

seaweeds (Gracilaria bursa-pastoris, Ulva rigida, and Gracilaria cornea) up to 10% in the diets

for European sea bass Dicentrarchus labrax did not affect the APD and ADMD of the test diets.

Moreover, Marinho et al. (2013) documented that utilization of Ulva spp up to 20% as a

replacement for 0-20% FM did not influence the APD and AED of the test diets for Nila tilapia

Oreochromis niloticus. In addition, Cyrus et al. (2015) summarized that 5% inclusion of Ulva

spp in the diets for sea urchin Tripneustes gratilla significantly improved APD, whereas

significantly reduced APD and AED were determined when Ulva spp was supplemented at 15%.

4.3. Growth performance and survival

In trial 1, UM1 can be included at 6.35% as a replacement for 2% FM without

compromising WG and FCR in a reference diet contained 10% FM. However, significant

reductions in WG and increment in FCR were determined when UM1 was supplemented from

12.7% to 25.4% as a replacement for 4% to 8% FM. Regression results demonstrate WG of

shrimp decreases as the inclusion level of UM1 increases, whereas FCR increases with

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enhancing UM1 levels (Figure 1a and 1b). Similarly, many studies demonstrated that low

inclusion levels (≤ 5%) of seaweed meals generally did not result in poor growth performance in

African catfish Clarias gariepinus (Abdel-Warith et al., 2016, Al-Asgah et al., 2016), European

sea bass (Valente et al., 2006), gilthead seabream Sparus aurata (Emre et al., 2013), Nile tilapia

(Marinho et al., 2013, Güroy et al., 2007, Valente et al., 2016), red tilapia Oreochromis Sp. (El-

Tawil, 2010), Pacific white shrimp (Rodríguez-González et al., 2014, Cárdenas et al., 2015), and

rainbow trout (Soler-Vila et al., 2009, Güroy et al., 2013).

However, the results of the moderate and high inclusion levels of seaweeds meals are

somewhat inconsistent. Rodríguez-González et al. (2014) indicated that dietary supplementation

of Ulva lactuca meal at both 10% and 15% as a substitution for FM significantly reduced the

WG of Pacific white shrimp, whereas shrimp fed with diets contained 10% and 15% Gracilaria

parvispora meal did not exhibit growth depression. Furthermore, Felix and Brindo (2014)

reported that dietary inclusion of raw Ulva lactuca meal at 10%, 20%, and 30% resulted in

depressed growth performance in giant freshwater prawn Macrobrachium rosenbergii,

nevertheless the supplementation of fermented Ulva lactuca meal at the same levels did not

affect the growth response. Moreover, Güroy et al. (2013) indicated that no difference in terms

of growth performance was determined when 10% raw or autoclaved Ulva rigida meal were

supplemented in the diet as a supplement for rainbow trout. However, significant reductions in

WG was detected in rainbow trout fed with diets contained 10% Ulva lactuca and Enteromorpha

linza meal (Yildirim et al., 2009). In addition, several studies demonstrated that Nile tilapia fed

with diets supplemented with Ulva spp. (a mixture of Ulva rigida and Ulva lactuca) or Ulva

rigida meal up to 10% did not exhibit difference with regards to the growth response, whereas

significant reduced WG was observed when inclusion level of those seaweed meals rose to 15%

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(Güroy et al., 2007, Marinho et al., 2013, Valente et al., 2016). Another two publications also

confirmed that dietary inclusion of Ulva lactuca and Gracilaria arcuata meals at 9% and 13.5%

significantly reduced the WG of African catfish and increased FCR (Abdel-Warith et al., 2016,

Al-Asgah et al., 2016). Variations among these researches could be attributed to the utilization of

different kinds of seaweed meal species and aquatic animal species as well as the over-

formulation of reference diet. The result might be masked as the reference diets in some of the

studies list above contained a high FM content resulting in over satisfaction of the nutrient

requirement of the target animals. As the protein, lipid, phosphorus, and amino acids

compositions are balanced in trial 1 (Table 4b), nutrient imbalances should be not counted as the

problem caused growth depression. The potential problems resulted in the reduced growth of

shrimp in trial 1 might be palatability shifts due to the reduced FM levels and the low nutrients

availability and high mineral concentrations in UM1.

As the replacement of FM results in shifts in numerous nutrients as well as possible

palatability changes of the diet, we chose to shift the nutrition model to replace a graded level of

SBM and compare the effects of three batches Ulva meal (UM1, 2, and 3) at high inclusion

levels in the trial 2. To compared the variations of Ulva meal from the three batches (UM1-3),

additional two diets (T2D8 and T2D9) basically formulated by using high levels of UM3 and

UM1, respectively, to replace the same amount of SBM as T2D5 which supplemented with UM2.

Results indicated that there was a decreasing trend of WG as the UM2 inclusion level increased

(Figure 2a). UM2 can be included up to 10% in the shrimp diet without causing growth

depression, whereas significant reduction in WG was determined when more than 15% UM2 was

supplemented in the diet. This phenomenon has been confirmed by trial 1 and multiple studies

cited above. Besides reduced growth performance, there was a decreasing trend of survival when

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more than 20% UM2 was included in the diet (Figure 2c). Significant reduction in survival was

detected at 25% and 30% inclusion level. Similarly, a number of studies have demonstrated that

low and moderate inclusion levels (≤20%) of various seaweed meals had no effects on the

survival of a variety of fish or shrimp species including Nile tilapia (Güroy et al., 2007), Pacific

white shrimp (Rodríguez-González et al., 2014), rainbow trout (Soler-Vila et al., 2009, Yildirim

et al., 2009), and red tilapia (El-Tawil, 2010). By contrast, Felix and Brindo (2014) reported that

survival of freshwater prawn was not affected when Ulva lactuca was used up to 30%. In

addition, Al-Asgah et al. (2016) documented that dietary Gracilaria arcuata meal inclusion up

to 30% did not affect the survival of African catfish. The different results were associated with

both seaweed and aquatic animal species. Shrimp fed with UM1 (T2D9) and UM3 (T2D8)

replacing the same levels of SBM as UM2 (T2D5) exhibited higher WG and lower FCR clearly

demonstrating differences across batches. Although UM2 produced the poorest result, UM3 also

resulted in significant reduction in growth.

Given the balanced nutrient composition of the test diets (Table 5b) and identical FM

level across all the treatments, nutrient imbalances and palatability shift can be eliminated as the

problems caused growth depression. The diet supplemented with UM1 (T2D9) contained highest

mineral contents among the test diets, whereas the shrimp fed this diet performed the best

compared to those fed with UM2 and UM3 replacing the same levels of SBM, which pointed out

high mineral composition was not the problem led to the reduced growth and survival. Low

nutrient availability of Ulva meal should be one of the potential problem result in growth

reduction, which can be mediated by formulating diets on a digestible basis.

To elucidate if digestible protein was limiting growth, a third trial was initiated for which

feeds were formulated on a digestible protein basis. In trial 3, four diets utilized high levels of

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Ulva meal from three batches to replace SBM on the digestible basis. Since methionine and

lysine are typically the two most limiting amino acids in shrimp feeds, they are also balanced on

the digestible basis. Results indicated that no significant differences in terms of WG, FCR, and

survival were detected in the diets contained UM1, however, significantly reduced growth and

survival as well as increased FCR were determined when shrimp fed with the diet contained

UM2. With regards to the supplementation of UM3, no differences were detected in WG and

FCR, however, survival was significantly reduced. The same trend for the WG was observed in

trial 2, in which reduced growth and survival as well as increased FCR were also observed for

shrimp fed with the diet supplemented with UM2. Although UM1 contained the highest level of

minerals among the three batches of Ulva meal (UM1-3), shrimp fed with UM1 performed the

best in terms of WG, FCR and survival, which also help to exclude minerals as one of the

problem. There must be some other factors affecting the growth of shrimp. The limited reduction

in performance of shrimp offered high levels of UM1 and UM3 indicated that part of the

problem is probably due to low digestibility of Ulva meal. However, this did not solve the

problem for UM2 which has both poor survival and growth. One theory that has been advanced

is that the Ulva sp. contains certain anti-nutritional factors or secretes certain secondary

poisonous metabolites which are detrimental for shrimp. However, identification of the specific

ANF or secondary poisonous metabolites was not available and beyond the scope of the current

study.

As the protein content of seaweed is tied to culture conditions, the high protein content of

Ulva meal can be achieved by culturing Ulva meal in water contains high nitrogen. The trial 4

evaluated the fourth batch Ulva meal which exhibited higher protein content (38.16%) than

UM1-3 as a replacement for FM and SBM. In general, there was a decreasing trend of WG and

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survival as the inclusion levels of UM4 increased (Figure 3a and 3d), which was in accordance

with previous trials. Shrimp fed with diets contained UM4 exhibited significantly reduced WG as

inclusion level of UM4 increases. Survival was significantly reduced when 9.5% UM4 was

incorporated in the diet to replace 6% FM.

4.4. Body compositions

In the current study, there was a consistently decreasing trend of whole body lipid content

as inclusion rates of Ulva meal increased in trial 1, 2, and 4. Significant difference in lipid

content was detected when UM1-3 were supplemented at more than 6.35, 10, and 9.5%,

respectively, in trial 1, 2, and 4. Similarly, Al-Asgah et al. (2016) recorded lipid content of

carcass was significantly reduced when more than 20% Gracilaria arcuata meal was

supplemented in the diets for African catfish. However, other authors did not report differences

in lipid content of whole body (Abdel-Warith et al., 2016, Emre et al., 2013, Felix and Brindo,

2014, Güroy et al., 2013, Güroy et al., 2007, Marinho et al., 2013, Rodríguez-González et al.,

2014, Soler-Vila et al., 2009, Valente et al., 2016, Valente et al., 2006, Yildirim et al., 2009).

The significantly reduced lipid content in the present study would be a result of comparatively

lower energy availability in the Ulva meal in contrast with the that in the FM and SBM.

Among the first three batches of Ulva meal, shrimp fed with UM2 exhibited significantly

lower lipid content of whole body compared to those fed with diets contained UM1 and UM3

replacing the same levels of SBM in trial 2. Similarly, shrimp fed with the diet contained UM2

formulating on the digestible protein basis exhibited significantly lower lipid content than those

fed with diets contain UM1 and UM3 in trial 3. The relatively lower lipid content in shrimp fed

with UM2 would be attributed to the significantly lower energy digestibility in UM2.

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Protein content was significantly improved when more than 10% UM2 was included in

the diets in trial 2. Similarly, in trial 4, significantly improved protein content was detected when

24% UM4 was incorporated in the diet. However, no significantly differences were observed in

the protein content in trial 1 and trial 3. A number of studies did not observe differences in the

protein content of whole body in a variety of aquatic animals (Emre et al., 2013, Felix and

Brindo, 2014, Güroy et al., 2013, Güroy et al., 2007, Valente et al., 2016, Valente et al., 2006,

Yildirim et al., 2009). By contrast, improvements in protein content were detected in African

catfish (Al-Asgah et al., 2016) and rainbow trout (Soler-Vila et al., 2009) when they are fed with

diets contained seaweed meals. The significantly improved protein content in the present study

might be an indirectly response to the dramatically reduced lipid content of shrimp body. As a

result of the enhanced protein content in trial 2, total amino acids and most individual amino

acids concentrations in shrimp body were improved in the treatments supplemented with Ulva

meal. Significant improvements were detected in the contents of arginine, cysteine, glycine,

histidine, lysine, methionine, and phenylalanine. In trial 3, no differences were determined in

total amino acids and most of individual amino acids except methionine contents in shrimp body,

which was in accordance with the unaffected protein content.

4.5. Protein and amino acid retention

In the present study, there were clearly decreasing trends of protein retention as Ulva

meal levels increased in trial 1, 2, and 4. Protein retention was determined by a number of factors

including dietary protein levels, feed offered, final weight, and initial weight of animals as well

as the final and initial protein content of animals (Halver and Hardy, 2002). In trial 1, 2, and 4,

the diets were balanced on an isonitrogenous basis. No difference was detected in the dietary

194

protein levels, feed offered and initial weight of shrimp. Although in trial 2 and trial 4, shrimp

fed with diets contained Ulva meal exhibited higher protein content of whole body, the improved

protein content cannot counteract the negative effects resulted by the significantly reduced

growth. Consequently, the significantly reduced protein retention in trial 1, 2, and 4 followed the

trends of decreased final mean weight as inclusion rates of Ulva meal enhanced. Similarly, a

number of studies have documented negative effects of seaweed meals on the protein retention in

Nile tilapia (Marinho et al., 2013), rainbow trout (Güroy et al., 2013, Soler-Vila et al., 2009,

Yildirim et al., 2009) and European sea bass (Valente et al., 2006).In trial 2, the dramatically

depressed protein retention translated to the significantly reduced amino acids retention. In

general, total amino acids and individual amino acid retention followed the decreasing trend of

protein retention as UM2 inclusion rate increased.

In trial 3, protein retention was significantly reduced when shrimp fed with diets

contained UM2 and UM3 compared to those fed with the reference diet. Among the diets

contained three batches of Ulva meal, shrimp fed diets contained UM2 performed the worst with

regards to protein retention compared to those fed with the diets supplemented with UM1 and

UM3. The test diets in trial 2 were formulated on the digestible basis. As protein digestibility of

Ulva meal is lower than that of SBM for which it substituted, more Ulva meal were included in

the diets resulting in higher protein contents in the diets supplemented with Ulva meal than the

reference diet. No differences were detected in feed offered, initial weight of shrimp, and final

and initial protein content of shrimp body. The reduced protein retention would be attributed to

the reduced final mean weight. There were reasonable correspondences between the amino acids

retention and protein retention. Total amino acids and individual amino acids retention in the

treatment contained UM2 were significantly depressed than other treatments.

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5. Conclusions

Under the reported conditions of this study, the results demonstrated a clear depressing in

the growth of shrimp when fish meal or soybean meal was replaced by Ulva meal. Among the

first three batches of Ulva meal (UM1-3), the second batch Ulva meal produced the worst result.

The low nutrient availability of Ulva meal in contrast with fish meal and soybean meal would be

part of the problems resulting in the growth depression. Other possible reasons which are beyond

the scope of this project but would include anti-nutrients present in the algae.

196

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204

Table 1 Proximate composition1, phosphorus content1, and amino acid profile1 of the primary protein sources used in diet formulations.

Composition (% as is)

Ulva meal 1

Ulva meal 2

Ulva meal 3

Ulva meal 4 Fish meal Soybean

meal Crude Protein 20.64 27.24 26.80 38.16 62.78 44.89 Moisture 8.89 13.74 11.19 8.41 7.99 10.97 Crude Fat 0.53 0.12 0.42 0.10 10.56 3.78 Crude Fiber 5.17 2.93 4.07 5.57 0.00 3.20 Ash 46.01 22.18 20.31 13.49 18.75 6.67 Phosphorus 0.43 0.30 - 0.42 3.15 0.66 Alanine 1.64 2.03 1.89 2.68 3.91 2.04 Arginine 0.99 1.39 1.01 1.77 3.68 3.35 Aspartic Acid 2.12 2.67 3.23 3.46 5.34 5.10 Cysteine 0.34 0.39 0.46 0.49 0.47 0.62 Glutamic Acid 2.02 2.59 3.02 3.35 7.47 8.24 Glycine 1.17 1.59 1.29 2.00 4.88 2.04 Histidine 0.25 0.40 0.22 0.45 1.63 1.2 Hydroxylysine 0.17 0.12 0.10 0.21 0.2 0.05 Hydroxyproline 0.2 0.30 0.38 0.35 1.03 0.05 Isoleucine 0.8 1.06 0.92 1.39 2.42 2.17 Leucine 1.22 1.87 1.50 2.43 4.21 3.57 Lysine 0.95 1.22 0.82 1.51 4.67 3.06 Methionine 0.26 0.44 0.46 0.63 1.61 0.66 Phenylalanine 0.98 1.37 1.16 1.78 2.39 2.35 Proline 0.76 1.17 1.02 1.50 3.08 2.39 Serine 0.91 1.05 0.93 1.47 2.11 1.90 Taurine 0.15 0.18 0.18 0.18 0.73 0.13 Threonine 0.94 1.17 1.13 1.56 2.41 1.75 Tryptophan 0.16 0.20 0.22 0.266 0.62 0.62 Tyrosine 0.48 0.77 0.49 0.94 1.67 1.64 Valine 1.17 1.56 1.40 2.13 2.99 2.34 1 Diets were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA).

205

Table 2 Mineral composition1 of the three batches Ulva meal (UM1, UM2, and UM3) utilized in feed formulations.

Minerals UM1 UM2 UM3 Quantity elements (% as is) Calcium 2.29 0.49 0.36 Potassium 1.99 2.21 3.9 Magnesium 2.57 2.93 1.11 Sodium 4.79 1.63 2.82 Phosphorus 0.4 0.32 0.31 Sulfur 3.46 4.54 2.96 Trace elements (mg kg-1 as is) Aluminum 4173.2 380.5 31.7 Arsenic 1.6 1.3 0.6 Boron 76.2 38.8 70.0 Barium 13.8 2.6 1.6 Cadmium 50.4 8.3 9.6 Cobalt 3.0 0.8 0.6 Chromium 9.7 1.8 0.7 Copper 26.5 17.5 56.9 Iron 9086.7 581.6 70.0 Manganese 112.4 21.1 17.8 Nickel 7.7 2.1 3.3 Lead 10.8 2.0 1.2 Selenium 5.3 3.9 2.8 Silicon 70.3 68.4 16.4 Zinc 63.1 34.6 18.9 Zirconium 1.0 1.0 1.0 1 Three batches Ulva meal were analyzed at Auburn University, Soils Laboratory (Auburn, AL, USA)

206

Table 3 Proximate1 and mineral1 composition of Ulva meal 2 (UM2) utilized in trial 2 and 3 collected from seven different dates.

Proximate composition (% as is)

Dates (2015)

7/21 7/30 8/16 8/20 8/23 8/25 8/30

Moisture 83.99 87.14 85.59 82.78 83.32 82.44 85.07

Crude protein 28.2 19.4 29 28.3 27.3 28 26.9

Crude fat 0.46 n.d. 0.2 n.d. n.d. 0.62 n.d.

Fiber 10 13.9 13.4 10.9 11.3 10.5 10.5

Ash 17.3 39.2 19.8 15.6 17.1 18.4 20.9

Quantity elements (% as is)

Calcium 0.42 2.01 0.86 0.47 0.44 0.42 0.46

Magnesium 3.12 3.07 3 3.25 3.21 3.25 3.12

Phosphorus 0.32 0.37 0.38 0.35 0.31 0.31 0.31

Potassium 2.71 2.26 1.82 2.2 1.95 2.31 2.49

Sodium 1.26 2.74 1.46 0.89 1.55 1.96 2.05

Sulfur 4.38 3.64 3.78 4.19 4.16 4.33 4.24

Trace elements (mg kg-1 as is)

Copper (ppm) 7.7 28.2 11 7.8 7.6 8.9 8.8

Iron (ppm) 331 6780 2040 424 450 356 510

Manganese (ppm) 21.1 99.2 47 22.9 22.2 21.9 24.3

Zinc (ppm) 37.6 79.3 64 49 38.4 38.9 38.8 1Analyses conducted by Midwest Laboratories (Omaha, NE, USA). n.d.: not detected.

207

Table 4a Formulation of test diets designed to evaluate Ulva meal 1 (UM1) as a replacement for fish meal on an iso-nitrogenous basis (Trial 1).

Ingredient (% as is) T1D1 T1D2 T1D3 T1D4 T1D5 Fish meal1 10.00 8.00 6.00 4.00 2.00 Soybean meal2 48.70 48.70 48.70 48.70 48.70 Corn protein concentrate3 8.00 8.00 8.00 8.00 8.00 Ulva meal 110

0.00 6.35 12.70 19.05 25.40 Fish oil2 5.65 5.75 5.86 5.97 6.07 Trace mineral premix5 0.50 0.50 0.50 0.50 0.50 Vitamin premix6 1.80 1.80 1.80 1.80 1.80 Choline chloride4 0.20 0.20 0.20 0.20 0.20 Stay C7 0.10 0.10 0.10 0.10 0.10 Mono-dicalcium phosphate8 1.62 1.90 2.15 2.40 2.65 Lecithin9 1.00 1.00 1.00 1.00 1.00 Cholesterol4 0.05 0.05 0.05 0.05 0.05 Corn starch4 22.54 17.78 13.05 8.31 3.58 1 Omega Protein Inc., Huston, TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5Trace mineral premix (g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA. 10 Ulva meal first batch experimentally produced.

208

Table 4b Proximate composition1 and amino acid profile1 of the test diets used in the trial 1.

Composition (% as is) T1D1 T1D2 T1D3 T1D4 T1D5

Ulva levels (%) 0 6.35 12.70 19.05 25.40

Crude Protein 36.83 36.52 36.60 36.28 35.65

Moisture 5.46 6.56 5.12 7.15 8.70

Crude Fat 10.09 8.94 9.06 8.22 7.51

Crude Fiber 2.92 3.08 3.48 3.22 3.33

Ash 6.54 8.92 11.80 14.40 16.58

Alanine 2.03 2.00 2.08 2.08 2.04

Arginine 2.24 2.21 2.23 2.19 2.14

Aspartic Acid 3.56 3.53 3.62 3.58 3.53

Cysteine 0.48 0.47 0.48 0.49 0.49

Glutamic Acid 6.39 6.18 6.32 6.11 6.01

Glycine 1.65 1.63 1.63 1.61 1.55

Histidine 0.92 0.89 0.89 0.85 0.82

Hydroxylysine 0.04 0.06 0.08 0.09 0.06

Hydroxyproline 0.13 0.12 0.11 0.10 0.09

Isoleucine 1.68 1.64 1.70 1.68 1.6

Leucine 3.43 3.29 3.41 3.34 3.21

Lysine 2.13 2.08 2.06 1.99 1.91

Methionine 0.66 0.64 0.63 0.62 0.63

Phenylalanine 1.85 1.86 1.94 1.92 1.81

Proline 2.09 2.08 2.12 2.09 1.98

Serine 1.51 1.50 1.53 1.47 1.51

Taurine 0.18 0.17 0.16 0.14 0.13

Threonine 1.36 1.35 1.38 1.37 1.35

Tryptophan 0.42 0.39 0.39 0.39 0.37

Tyrosine 1.45 1.44 1.49 1.47 1.39

Valine 1.78 1.77 1.82 1.80 1.76 1 Diets were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA).

20

9

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210

Table 4d Proximate3 analysis of whole shrimp body offered varying Ulva meal 1 levels (0, 6.35, 12.70, 19.05, and 25.40%) as a replacement of fish meal over a six-week growth trial (Trial 1).

Diet Ulva Levels (%) Moisture (%) Crude protein2 (%) Crude lipid2 (%)

T1D1 0 76.88 72.77 8.04a

T1D2 6.35 76.29 73.63 6.12b

T1D3 12.70 76.99 74.27 5.73b

T1D4 19.05 76.37 72.83 5.99b

T1D5 25.40 76.83 74.11 5.09b

P-value 0.7933 0.2576 0.0006

PSE1 0.1340 0.2240 0.1613 1 Pooled standard error. 2 Dry weight basis. 3 Body samples were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA). Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

211

Table 5a Formulation of test diets designed to evaluate three batches of Ulva meal (UM1, UM2, and UM3) as a replacement for soybean meal on an iso-nitrogenous basis (Trial 2).

Ingredient (% As is basis) T2D1 T2D2 T2D3 T2D4 T2D5 T2D6 T2D7 T2D8 T2D9

Fish meal1 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

Soybean meal2 55.55 52.55 49.60 46.60 43.75 40.80 37.80 43.75 43.75 Corn protein concentrate3 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

Ulva meal 211 0.00 5.00 10.00 15.00 20.00 25.00 30.00 0.00 0.00

Ulva meal 311 0.00 0.00 0.00 0.00 0.00 0.00 0.00 23.60 0.00

Ulva meal 111 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 26.30

Fish oil2 5.84 5.88 5.92 5.96 6.00 6.04 6.08 6.00 5.89

Trace mineral premix5 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50

Vitamin premix6 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80

Choline chloride4 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20

Stay C7 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Mono-dicalcium phosphate8 1.85 1.85 1.90 1.90 1.95 1.95 2.00 1.90 1.70

Lecithin9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Cholesterol4 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Methionine10 0.07 0.06 0.06 0.06 0.05 0.05 0.04 0.03 0.07

Corn starch4 15.04 13.01 10.87 8.83 6.60 4.51 2.43 3.07 0.64 1 Omega Protein Inc., Huston TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5 Trace mineral premix (g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA. 10 Aldrich-Sigma, St. Louis, MO, USA.

212

11 Three batches of Ulva meal experimentally produced.

213

Table 5b Proximate composition1, phosphorus content1, and amino acid profile1 of the test diets used in the trial 2.

Composition1 (% as is)

T2D1 T2D2 T2D3 T2D4 T2D5 T2D6 T2D7 T2D8 T2D9

Ulva levels (%) 0 5 10 15 20 25 30 23.6 26.3 Crude Protein 36.46 36.67 35.91 36.78 36.64 36.69 37.46 37.08 36.34

Moisture 7.32 7.92 9.44 7.56 8.29 8.03 6.46 7.74 8.48 Crude Fat 10.02 8.49 8.68 9.71 8.90 8.28 6.29 6.35 7.01

Crude Fiber 3.54 3.43 3.65 3.64 3.79 4.10 4.09 3.86 3.86 Ash 6.49 7.61 8.47 9.02 10.17 10.47 11.48 10.56 16.77

Phosphorus 0.99 1.01 1.00 1.03 1.01 1.06 1.02 1.00 1.02 Alanine 1.86 1.90 1.91 2.00 2.11 2.05 2.18 2.03 1.99 Arginine 2.30 2.26 2.20 2.22 2.19 2.18 2.24 2.12 2.12

Aspartic Acid 3.68 3.62 3.56 3.62 3.61 3.60 3.68 3.72 3.50 Cysteine 0.50 0.47 0.47 0.48 0.48 0.47 0.47 0.50 0.47

Glutamic Acid 6.68 6.48 6.25 6.29 6.26 5.98 6.07 6.14 5.95 Glycine 1.64 1.68 1.67 1.69 1.84 1.77 1.86 1.73 1.62

Histidine 0.91 0.88 0.85 0.86 0.84 0.82 0.84 0.79 0.80 Hydroxylysine 0.05 0.05 0.05 0.06 0.07 0.07 0.07 0.06 0.07 Hydroxyproline 0.11 0.10 0.11 0.12 0.18 0.37 0.16 0.22 0.17

Isoleucine 1.64 1.60 1.56 1.60 1.58 1.57 1.61 1.56 1.54 Leucine 3.22 3.14 3.09 3.16 3.20 3.08 3.22 3.03 3.00 Lysine 2.04 1.99 1.95 1.95 1.91 1.92 1.94 1.86 1.86

Methionine 0.70 0.67 0.66 0.67 0.68 0.66 0.67 0.63 0.61 Phenylalanine 1.85 1.82 1.80 1.84 1.86 1.82 1.89 1.79 1.76

Proline 2.09 1.97 2.06 1.96 2.13 2.02 1.99 2.03 2.00 Serine 1.53 1.53 1.47 1.52 1.51 1.49 1.54 1.45 1.46

Taurine 0.14 0.16 0.15 0.15 0.15 0.16 0.17 0.17 0.14 Threonine 1.35 1.35 1.34 1.37 1.38 1.39 1.44 1.37 1.34

Tryptophan 0.50 0.50 0.47 0.48 0.48 0.47 0.49 0.44 0.45 Tyrosine 1.19 1.19 1.14 1.21 1.19 1.17 1.21 1.10 1.13 Valine 1.83 1.81 1.80 1.88 1.88 1.86 1.91 1.84 1.80

1 Diets were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA).

214

Table 5c Mineral profile1 of the test diets used in the trial 2. Mineral T2D1 T2D2 T2D3 T2D4 T2D5 T2D6 T2D7 T2D8 T2D9 Quantity elements (g kg-1) Calcium 8.5 9.7 9.0 9.5 9.1 8.9 9.5 9.3 13.3 Potassium 12.7 13.5 13.7 14.6 14.9 14.9 15.9 18.8 15.5 Magnesium 1.9 3.4 4.7 6.2 7.5 8.7 9.6 4.8 8.3 Sodium 0.8 1.7 2.4 3.3 4.1 4.8 5.7 7.4 14.0 Phosphorus 10.3 10.8 10.2 10.7 10.3 9.9 10.4 10.5 8.3 Sulfur 3.8 5.9 7.7 10.0 11.8 13.6 16.2 10.4 12.0 Trace elements (mg kg-1) Aluminum 97.8 119.5 133.1 160.8 173.8 185.6 199.1 99.7 1175.4 Arsenic 0.7 0.6 0.4 0.6 0.8 0.9 0.4 0.4 1.0 Boron 17.6 18.6 19.3 20.5 21.5 21.5 23.8 29.7 34.5 Barium 5.1 5.3 5.7 5.1 5.0 4.5 4.6 4.4 8.2 Cadmium 4.5 0.9 13.2 1.2 14.3 12.6 6.5 1.4 13.7 Cobalt 1.1 1.3 1.2 1.2 1.2 1.0 1.1 1.0 1.7 Chromium 1.0 1.0 1.1 1.1 1.1 1.1 1.1 0.9 3.6 Copper 39.4 110.3 21.0 22.9 18.6 23.9 27.1 28.8 23.9 Iron 59.2 43.8 69.4 74.6 66.9 74.4 66.2 48.7 904.5 Manganese 34.2 35.4 34.5 33.6 33.5 32.7 34.1 33.0 61.4 Molybdenum 3.9 4.0 3.4 3.3 3.5 2.5 2.7 3.1 0.1 Nickel 3.1 3.2 3.1 2.6 2.7 2.4 2.2 2.7 4.5 Lead 1.0 1.3 1.7 0.5 1.4 0.5 0.6 0.2 4.0 Selenium 3.3 4.6 2.3 3.1 3.8 4.4 4.8 4.5 4.2 Silicon 57.9 81.0 107.0 120.8 119.9 131.7 131.4 59.9 61.8 Zinc 158.1 165.1 145.8 145.8 135.6 155.4 153.9 152.6 174.1 Zirconium 0.9 1.0 0.9 0.9 1.0 0.9 0.9 0.9 0.7 1 Diets were analyzed at Auburn University, Soils Laboratory (Auburn, AL, USA)

215

Table 5d Growth performance of juvenile Pacific white shrimp (0.24±0.01g) offered diets with

different levels of three batches Ulva meal (UM1, UM2, and UM3) for five weeks (Trial 2).

Diet Ulva levels

(%) Final

Biomass (g) Final Mean Weight (g) WG3 (%) FCR2 Survival

(%) T2D1 0 43.31a 4.55a 1734.21a 1.46b 95.0a

T2D2 5 36.19ab 3.70ab 1398.22ab 1.83ab 97.5a

T2D3 10 28.40bc 3.25ab 1241.46ab 2.23ab 87.5ab

T2D4 15 23.89cd 2.58b 948.74b 2.82ab 92.5a

T2D5 20 18.98cd 2.53b 990.26b 2.96ab 75.0ab

T2D6 25 16.34cd 2.56b 943.46b 3.53a 67.5b

T2D7 30 15.50d 2.45b 864.67b 3.37ab 65.0b

T2D8 23.6 26.14bcd 2.96b 1131.07b 2.61ab 87.5ab

T2D9 26.3 27.10bcd 3.09b 1226.92ab 2.36ab 87.5ab

P-value <0.0001 0.0002 0.0008 0.0201 0.0006

PSE1 1.2872 0.1392 61.8604 0.2020 2.6131 1 Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - Initial weight) / Initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

21

6

Tab

le 5

e Pr

oxim

ate

com

posi

tion2 a

nd a

min

o ac

id p

rofil

e2 of w

hole

shr

imp

body

off

ered

die

ts c

onta

in d

iffer

ent l

evel

s of

thre

e ba

tche

s U

lva

mea

l (U

M1,

UM

2, a

nd U

M3)

leve

ls in

tria

l 2.

Die

t T 2

D1

T 2D

2 T 2

D3

T 2D

4 T 2

D5

T 2D

6 T 2

D7

T 2D

8 T 2

D9

PSE1

P-va

lue

Adj

ust

P- valu

e U

lva

leve

ls (%

) 0

5 10

15

20

25

30

23

.6

26.3

Moi

stur

e 75

6.6

780.

2 76

0.5

773.

3 76

8.3

771.

6 78

0.5

770.

4 76

7.6

0.32

04

0.19

25

0.33

00

Prot

ein

725.

6d 72

8.1cd

74

3.3ab

c 75

1.8ab

75

5.0a

747.

7ab

754.

3a 74

1.5ab

cd

736.

2bcd

0.18

02

<0.0

001

0.00

01

Lipi

d 77

.2a

62.2

ab

45.4

bc

27.5

d 29

.0cd

29

.7cd

22

.0d

47.4

b 52

.7b

0.17

79

<0.0

001

0.00

01

Ala

nine

4.

30

4.50

4.

40

4.37

4.

36

4.20

4.

28

4.36

4.

34

0.02

56

0.02

98

0.06

50

Arg

inin

e 4.

98c

4.91

c 4.

95c

5.21

abc

5.22

abc

5.43

ab

5.51

a 5.

13bc

5.

18ab

c 0.

0390

<0

.000

1 0.

0003

A

spar

tic A

cid

6.68

6.

85

6.87

6.

93

6.87

6.

79

6.85

6.

89

6.85

0.

037

0.52

57

0.63

09

Cys

tein

e 0.

57c

0.58

c 0.

60bc

0.

62ab

0.

62ab

0.

62ab

0.

64a

0.60

abc

0.60

abc

0.00

37

<0.0

001

0.00

02

Glu

tam

ic A

cid

9.96

10

.10

10.2

1 10

.24

10.2

5 10

.09

10.0

6 10

.11

10.2

7 0.

0694

0.

7825

0.

8412

G

lyci

ne

4.53

c 4.

68bc

4.

85bc

5.

28ab

c 5.

10ab

c 5.

45ab

5.

68a

4.98

abc

4.98

abc

0.08

42

0.00

11

0.00

50

His

tidin

e 1.

48b

1.52

ab

1.58

a 1.

59a

1.60

a 1.

59a

1.55

ab

1.60

a 1.

56ab

0.

0103

0.

0031

0.

0105

H

ydro

xyly

sine

0.

14

0.13

0.

16

0.16

0.

16

0.14

0.

14

0.16

0.

15

0.00

07

0.39

39

0.52

52

Hyd

roxy

prol

ine

0.21

0.

20

0.20

0.

21

0.20

0.

21

0.20

0.

21

0.21

0.

0051

0.

9706

0.

9706

Is

oleu

cine

2.

87

2.90

2.

95

2.96

2.

96

2.93

2.

90

2.92

2.

91

0.01

29

0.24

23

0.36

35

Leuc

ine

4.80

4.

87

4.92

4.

96

4.99

4.

93

4.90

4.

92

4.90

0.

0227

0.

2309

0.

3635

Ly

sine

4.

72b

4.86

ab

4.92

ab

5.02

a 5.

05a

5.06

a 5.

06a

4.99

ab

4.94

ab

0.03

12

0.00

95

0.02

53

Met

hion

ine

1.40

b 1.

41b

1.42

b 1.

47ab

1.

46ab

1.

48ab

1.

52a

1.44

ab

1.46

ab

0.00

95

0.00

28

0.01

05

Phen

ylal

anin

e 3.

10b

3.12

b 3.

21ab

3.

24ab

3.

28a

3.20

ab

3.14

ab

3.23

ab

3.12

b 0.

0159

0.

0044

0.

0132

Pr

olin

e 3.

80

3.74

3.

85

3.73

3.

95

3.78

3.

55

3.65

3.

63

0.06

39

0.52

16

0.63

09

Serin

e 2.

29

2.34

2.

33

2.37

2.

37

2.35

2.

38

2.35

2.

42

0.02

37

0.80

62

0.84

12

21

7

Thre

onin

e 2.

64

2.68

2.

71

2.71

2.

74

2.68

2.

64

2.72

2.

70

0.01

36

0.15

40

0.28

44

Tryp

toph

an

0.84

0.

87

0.88

0.

90

0.89

0.

89

0.89

0.

89

0.88

0.

0054

0.

0272

0.

0650

Ty

rosi

ne

2.28

2.

23

2.50

2.

52

2.54

2.

48

2.16

2.

50

2.47

0.

0643

0.

3161

0.

4462

V

alin

e 4.

05

4.09

4.

07

4.09

4.

22

4.12

4.

12

4.11

4.

20

0.03

67

0.76

81

0.84

12

Tota

l 65

.61

66.5

5 67

.56

68.5

8 68

.82

68.4

1 68

.15

67.7

4 67

.71

0.32

62

0.03

73

0.07

46

1 Poo

led

stan

dard

err

or.

2 B

ody

sam

ples

wer

e an

alyz

ed a

t Uni

vers

ity o

f Mis

sour

i-Col

umbi

a, A

gric

ultu

re E

xper

imen

t Sta

tion

Che

mic

al L

abor

ator

y (C

olum

bia,

M

O, U

SA).

Val

ues w

ithin

a ro

w w

ith d

iffer

ent s

uper

scrip

ts a

re si

gnifi

cant

ly d

iffer

ent b

ased

on

Tuke

y’s m

ultip

le ra

nge

test

.

21

8

Tab

le 5

f Pro

tein

2 and

am

ino

acid

s3 rete

ntio

n ef

ficie

ncie

s of P

acifi

c w

hite

shrim

p of

fere

d va

ryin

g U

lva

mea

l 2 le

vels

die

ts in

tria

l 2 fo

r fiv

e w

eeks

.

Die

t T 2

D1

T 2D

2 T 2

D3

T 2D

4 T 2

D5

T 2D

6 T 2

D7

T 2D

8 T 2

D9

P-va

lue

PSE1

Adj

ust

P-va

lue

Ulv

a le

vels

(%)

0 5

10

15

20

25

30

23.6

26

.3

Prot

ein

36.4

a 26

.5ab

25

.6ab

19

.0b

18.1

b 17

.1b

15.3

b 21

.4b

22.9

b 1.

1928

<0

.000

1 0.

0002

A

lani

ne

42.3

a 31

.8ab

28

.5bc

20

.2bc

d 18

.1cd

17

.2cd

14

.8d

23.0

bcd

24.7

bcd

1.31

31

<0.0

001

<0.0

001

Arg

inin

e 39

.8a

29.2

ab

28.1

ab

21.9

b 21

.1b

21.1

b 19

.0b

26.1

b 27

.9ab

1.

3498

0.

0003

0.

0005

A

spar

tic A

cid

33.2

a 25

.3ab

23

.9ab

c 17

.7bc

16

.7bc

15

.9bc

14

.1c

19.9

bc

22.1

bc

1.11

08

<0.0

001

0.00

02

Cys

tein

e 20

.8a

16.5

ab

15.6

ab

11.9

b 11

.3b

11.0

b 10

.2b

12.8

b 14

.4ab

0.

7296

0.

0004

0.

0005

G

luta

mic

Aci

d 27

.3a

20.9

ab

20.3

ab

15.1

b 14

.4b

14.3

b 12

.6b

17.7

b 19

.6ab

0.

9825

0.

0004

0.

0005

G

lyci

ne

50.5

a 37

.3ab

36

.1ab

29

.1b

24.4

b 25

.5b

23.4

b 31

.0b

34.9

ab

1.63

81

<0.0

001

0.00

02

His

tidin

e 29

.7a

23.0

ab

23.0

ab

17.1

b 16

.7b

16.3

b 13

.9b

21.7

ab

21.9

ab

1.11

48

0.00

10

0.00

10

Hyd

roxy

lysi

ne

50.3

a 31

.6ab

c 38

.6ab

25

.2bc

20

.3bc

16

.6bc

14

.2c

29.3

abc

23.1

bc

2.42

99

0.00

04

0.00

05

Hyd

roxy

prol

ine

34.0

a 26

.6ab

22

.7bc

15

.9cd

9.

8de

4.9e

9.6de

10

.4de

14

.0d

0.82

58

<0.0

001

<0.0

001

Isol

euci

ne

32.1

a 24

.3ab

23

.4ab

c 17

.2bc

16

.5bc

15

.8bc

13

.7c

20.1

bc

21.3

bc

1.10

65

0.00

01

0.00

02

Leuc

ine

27.3

a 20

.7ab

19

.8ab

c 14

.5bc

13

.7bc

13

.5bc

11

.5c

17.4

bc

18.5

abc

0.94

49

<0.0

001

0.00

02

Lysi

ne

42.2

a 32

.7ab

31

.2ab

23

.8b

23.2

b 22

.2b

19.8

b 28

.7ab

30

.0ab

1.

5286

0.

0006

0.

0007

M

ethi

onin

e 36

.4a

28.2

ab

26.8

ab

20.4

b 18

.9b

18.9

b 17

.2b

24.5

ab

27.0

ab

1.34

55

0.00

05

0.00

06

Phen

ylal

anin

e 30

.6a

22.9

ab

22.1

abc

16.3

bc

15.5

bc

14.8

bc

12.6

c 19

.3bc

20

.0bc

1.

0502

<0

.000

1 0.

0002

Pr

olin

e 33

.3a

25.5

ab

23.3

abc

17.8

bc

16.4

bc

16.4

bc

13.6

c 19

.2bc

20

.5bc

1.

2344

0.

0002

0.

0004

Se

rine

27.4

a 20

.4ab

19

.6ab

14

.4b

13.8

b 13

.3b

11.8

b 17

.3b

18.8

ab

0.94

69

0.00

01

0.00

02

Thre

onin

e 35

.7a

26.5

ab

25.0

ab

18.3

bc

17.4

bc

16.3

bc

13.9

c 21

.3bc

22

.8bc

1.

1486

<0

.000

1 0.

0001

Tr

ypto

phan

30

.7a

23.0

ab

23.1

ab

17.3

b 16

.2b

15.9

b 13

.8b

21.7

ab

21.9

ab

1.10

80

0.00

04

0.00

05

Tyro

sine

35

.2a

24.7

abc

27.1

ab

19.4

bc

18.7

bc

17.9

bc

13.1

c 24

.4ab

c 24

.6ab

c 1.

4386

0.

0008

0.

0008

21

9

Val

ine

40.4

a 30

.1ab

28

.0ab

c 20

.2bc

19

.7bc

18

.7bc

16

.4c

24.0

bc

26.5

bc

1.36

55

<0.0

001

0.00

02

Tota

l 33

.5a

25.3

ab

24.2

ab

18.1

b 17

.0b

16.5

b 14

.5b

21.0

b 22

.7ab

1.

1483

0.

0001

0.

0002

1 P

oole

d st

anda

rd e

rror

. 2 Pr

otei

n re

tent

ion

effic

ienc

y =

(Fin

al w

eigh

t × F

inal

pro

tein

con

tent

) - (I

nitia

l wei

ght ×

Initi

al p

rote

in c

onte

nt) ×

100

/ Pr

otei

n of

fere

d.

3 A

min

o ac

ids

(AA

) re

tent

ion

effic

ienc

y =

(Fin

al w

eigh

t ×

Fina

l A

A c

onte

nt)

- (I

nitia

l w

eigh

t ×

Initi

al A

A c

onte

nt) ×

100

/ A

A

offe

red.

V

alue

s with

in a

row

with

diff

eren

t sup

ersc

ripts

are

sign

ifica

ntly

diff

eren

t bas

ed o

n Tu

key’

s mul

tiple

rang

e te

st.

220

Table 6a Formulation of test diets designed to evaluate Ulva meal 1, 2, and 3 as a replacement for soybean meal on a digestible protein basis (Trial 3).

Ingredient (% as is) T3D1 T3D2 T3D3 T3D4 Fish meal1 6.00 6.00 6.00 6.00 Soybean meal2 53.00 46.30 49.90 46.30 Corn protein concentrate3 8.00 8.00 8.00 8.00 Ulva meal 210

22.00 Ulva meal 110 25.00 Ulva meal 310 25.00 Fish oil2 5.92 5.98 5.85 5.91 Trace mineral premix5 0.50 0.50 0.50 0.50 Vitamin premix6 1.80 1.80 1.80 1.80 Choline chloride4 0.20 0.20 0.20 0.20 Stay C7 0.10 0.10 0.10 0.10 Mono-dicalcium phosphate8 2.50 2.50 2.50 2.50 Lecithin9 1.00 1.00 1.00 1.00 Cholesterol4 0.08 0.08 0.08 0.08 Methionine 0.05 0.04 0.04 0.04 Lyisine 0.00 0.07 0.00 0.11 Corn starch4 20.85 2.43 2.03 2.46 1 Omega Protein Inc., Huston TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5Trace mineral premix (g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA. 10 Three batches Ulva meal experimentally produced.

221

11 Aldrich-Sigma, St. Louis, MO, USA.

222

Table 6b Proximate composition1, mineral composition2, and amino acid profile1 of the test diets used in trial 3.

Composition T3D1 T3D2 T3D3 T3D4 Proximate composition (% as is) Crude protein 36.33 38.40 39.66 39.13 Moisture 7.15 7.59 8.93 8.34 Crude fat 9.39 9.03 9.01 8.68 Crude fiber 3.21 3.84 4.42 4.13 Ash 6.86 15.93 11.44 11.22 Quantity elements (% as is) Phosphorus 1.36 1.25 1.24 1.37 Sulfur 0.4 1.06 1.27 1.08 Potassium 1.33 1.73 1.65 2.13 Magnesium 0.18 0.76 0.86 0.52 Calcium 1.31 1.79 1.17 1.30 Trace elements (mg kg-1 as is) Sodium 0.1 1.16 0.51 0.77 Iron (ppm) 149 1240 286 169 Manganese (ppm) 40.1 71.6 39.1 40.1 Copper (ppm) 16.8 22.9 20.2 28.7 Zinc (ppm) 183 215 187 194 Amino acid profile (% as is) Alanine 1.87 2.15 2.24 2.14 Arginine 2.18 2.26 2.34 2.21 Aspartic Acid 3.44 3.66 3.78 3.79 Cysteine 0.48 0.49 0.50 0.51 Glutamic Acid 6.33 6.43 6.33 6.24 Glycine 1.56 1.69 1.82 1.68 Histidine 0.86 0.86 0.89 0.80 Isoleucine 1.60 1.70 1.71 1.65 Leucine 3.28 3.49 3.50 3.32 Lysine 2.01 2.03 2.16 2.05 Methionine 0.64 0.62 0.67 0.65 Phenylalanine 1.85 2.00 2.05 1.90 Proline 2.13 2.16 2.22 2.22

223

Serine 1.48 1.61 1.68 1.59 Taurine 0.16 0.13 0.14 0.16 Threonine 1.29 1.43 1.50 1.44 Tryptophan 0.47 0.48 0.45 0.44 Tyrosine 1.33 1.38 1.44 1.33 Valine 1.73 1.95 2.01 1.94 1 Proximate composition and amino acid profiles of test diets were analyzed at University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA). 2 Mineral composition was tested at Midwest Laboratories (Omaha, NE, USA).

224

Table 6c Growth performance of juvenile Pacific white shrimp L. vannamei (Initial weight 0.98g) offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal over six weeks (Trial 3).

Diet Final biomass (g) Final mean

weight (g) WG3 (%) FCR2 Survival (%)

T3D1 79.3a 8.4a 766.6a 1.64b 95.0a

T3D2 66.8a 7.8a 689.7a 1.87b 85.0ab

T3D3 30.6c 4.9b 397.3b 3.58a 62.5c

T3D4 57.3b 7.2a 618.1a 2.09b 80.0b

PSE1 1.7351 2.3457 18.1043 0.1167 1.5427

P-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 1 Pooled standard error. 2 FCR: Feed conversion ratio = Feed offered / (Final weight - Initial weight). 3 WG: Weight gain = (Final weight - Initial weight) / Initial weight × 100%. Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

225

Table 6d Proximate composition2 and amino acids profile2 of shrimp at the conclusion of a 6-week growth trial in which shrimp were offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal (Trial 3).

Diet T3D1 T3D2 T3D3 T3D4 PSE1 P-value Adjust P-value

Moisture 75.65b 76.32ab 77.88a 75.56b 0.2029 0.0054 0.0432 Protein 75.08 76.70 76.98 75.24 0.2774 0.0675 0.1800 Lipid 6.37a 5.04a 2.68b 5.31a 0.2479 0.0015 0.0180 Alanine 4.27 4.21 4.21 4.36 0.0416 0.5419 0.5612 Arginine 5.45 5.89 5.67 5.31 0.0543 0.0126 0.0756 Aspartic Acid 6.76 6.94 7.01 6.96 0.0440 0.2679 0.4559 Cysteine 0.60 0.63 0.64 0.61 0.0045 0.0387 0.1327 Glutamic Acid 10.18 10.51 10.47 10.40 0.0708 0.3940 0.4728 Glycine 5.01b 5.41ab 5.90a 5.07b 0.0962 0.0246 0.0984 Histidine 1.49 1.56 1.58 1.54 0.0156 0.2442 0.4508 Hydroxylysine 0.14 0.17 0.16 0.14 0.0067 0.2203 0.4406 Hydroxyproline 0.20 0.22 0.22 0.21 0.0034 0.3229 0.4559 Isoleucine 2.95 3.00 2.95 3.01 0.0150 0.3048 0.4559 Leucine 4.95 5.08 5.00 5.03 0.0264 0.3920 0.4728 Lysine 4.92 5.20 5.13 5.10 0.0281 0.0234 0.0984 Methionine 1.46b 1.58a 1.53a 1.53a 0.0071 0.0007 0.0168 Phenylalanine 3.16 3.28 3.26 3.19 0.0314 0.5019 0.5551 Proline 4.09 4.14 3.79 4.11 0.0750 0.3737 0.4728 Serine 2.35 2.42 2.47 2.38 0.0286 0.5088 0.5551 Threonine 2.62 2.71 2.73 2.71 0.0136 0.0453 0.1359 Tryptophan 0.87 0.88 0.88 0.89 0.0046 0.5612 0.5612 Tyrosine 2.51 2.59 2.59 2.36 0.0459 0.3004 0.4559 Valine 4.10 4.21 4.36 4.23 0.0322 0.0935 0.2040 Total 68.05 70.61 70.52 69.08 0.3708 0.0884 0.2040 1 Pooled standard error. 2 Body samples were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA). Values within a row with different superscripts are significantly different based on Tukey’s multiple range test.

226

Table 6e Protein2 and amino acid3 retention efficiency of Pacific white shrimp at the conclusion of a 6-week growth trial in which shrimp were offered diets formulated to partially replace soybean meal on a digestible protein basis with three different batches of Ulva meal (Trial 3).

Retention T3D1 T3D2 T3D3 T3D4 P-value PSE1 Adjust P-value

Protein 34.2a 29.4ab 14.4c 26.0b 0.8054 <0.0001 <0.0001 Alanine 37.5a 28.5a 13.6b 27.3a 0.6848 <0.0001 <0.0001 Arginine 41.5a 38.8b 18.2c 32.5b 1.1379 <0.0001 <0.0001 Aspartic Acid 32.6a 28.0a 13.9b 24.9a 0.7763 <0.0001 <0.0001 Cysteine 20.5a 18.9ab 9.6c 16.2b 0.5597 <0.0001 0.0001 Glutamic Acid 26.7a 24.2a 12.4b 22.6a 0.6799 <0.0001 <0.0001 Glycine 53.4a 47.9a 25.0b 41.3a 1.5060 <0.0001 0.0002 Histidine 28.6a 26.9a 13.3b 26.2a 0.8115 <0.0001 0.0001 Hydroxylysine 29.3ab 25.7b 11.2b 44.7a 2.1886 0.0015 0.0015 Hydroxyproline 16.9a 13.4b 7.6c 11.3b 0.3435 <0.0001 <0.0001 Isoleucine 30.5a 26.1a 12.9b 24.8a 0.6891 <0.0001 <0.0001 Leucine 25.0a 21.6a 10.7b 20.6a 0.5786 <0.0001 <0.0001 Lysine 40.7a 38.1a 18.0b 33.9a 0.9340 <0.0001 <0.0001 Methionine 38.1a 38.1a 17.3b 32.2a 0.9552 <0.0001 <0.0001 Phenylalanine 28.3a 24.3a 11.9b 22.7a 0.7167 <0.0001 <0.0001 Proline 32.4a 28.8a 13.2b 25.5a 0.8271 <0.0001 <0.0001 Serine 26.3a 22.2ab 11.0c 20.3b 0.6670 <0.0001 <0.0001 Threonine 33.6a 28.1ab 13.7c 25.5b 0.7495 <0.0001 <0.0001 Tryptophan 30.5a 27.2a 14.5b 27.3a 0.7670 <0.0001 <0.0001 Tyrosine 31.2a 27.7a 13.5b 23.8a 0.9244 0.0001 0.0001 Valine 39.4a 32.0ab 16.4c 29.7b 0.9798 <0.0001 <0.0001 Total 32.3a 28.4ab 14.1c 25.8b 0.7669 <0.0001 <0.0001 1 Pooled standard error. 2 Protein retention efficiency = (Final weight × Final protein content) - (Initial weight × Initial protein content) × 100 / Protein offered. 3 Amino acids retention efficiency = (Final weight × Final amino acids content) - (Initial weight × Initial amino acids content) × 100 / Amino acids offered. Values within a row with different superscripts are significantly different based on Tukey’s multiple range test.

227

Table 7a Formulation of test diets designed to evaluate Ulva meal 4 as a protein source in trial 4.

Ingredient (% as is) T4D1 T4D2 T4D3 T4D4 T4D5

Fish meal1 6.00 6.00 6.00 3.00 0.00

Soybean meal2 53.00 43.00 33.00 53.00 53.00

Corn protein concentrate3 8.00 8.00 8.00 8.00 8.00

Ulva meal 411 0.00 12.00 24.00 4.75 9.50

Fish oil2 5.92 6.05 6.18 6.19 6.45

Trace mineral premix5 0.50 0.50 0.50 0.50 0.50

Vitamin premix6 1.80 1.80 1.80 1.80 1.80

Choline chloride4 0.20 0.20 0.20 0.20 0.20

Stay C7 0.10 0.10 0.10 0.10 0.10

Mono-dicalcium phosphate8 2.50 2.60 2.60 2.90 3.10

Lecithin9 1.00 1.00 1.00 1.00 1.00

Cholesterol4 0.08 0.08 0.08 0.08 0.08

Lyisine 10 0.00 0.11 0.22 0.07 0.13

Methionine10 0.05 0.11 0.17 0.10 0.15

Corn starch4 20.85 18.45 16.15 18.31 15.99 1 Omega Protein Inc., Huston TX, USA. 2 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 3 Empyreal® 75, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA. 4 MP Biomedicals Inc., Solon, OH, USA. 5 Trace mineral premix (g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 6 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 J. T. Baker®, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. 9 The Solae Company, St. Louis, MO, USA. 10 Fourth batch Ulva meal experimentally produced. 11 Aldrich-Sigma, St. Louis, MO, USA.

228

Table 7b Proximate composition1, mineral composition2 and amino acid profile1 of the test diets used in trial 4.

Composition T4D1 T4D2 T4D3 T4D4 T4D5 Proximate composition (% as is) Crude protein 35.70 35.20 35.00 34.30 33.40 Moisture 8.70 9.93 10.2 9.89 10.22 Crude fat 6.71 8.37 8.65 8.03 8.21 Crude fiber 3.10 7.30 6.40 5.80 8.40 Ash 7.08 7.67 9.19 7.22 7.24 Quantity elements (% as is) Sulfur 0.40 0.74 1.19 0.56 0.72 Phosphorus 1.36 1.03 1.08 1.09 1.10 Potassium 1.33 1.14 1.20 1.24 1.35 Magnesium 0.18 0.40 0.66 0.29 0.40 Calcium 1.31 1.27 1.36 1.32 1.36 Sodium 0.10 0.23 0.40 0.13 0.17 Trace elements (mg kg-1) Iron 149 165 193 125 136 Manganese 40.1 50.9 54.9 54.4 59.9 Copper 16.8 16.7 15.2 16.3 16.1 Zinc 183 173 266 292 212 Amino acid profile (% as is) Alanine 1.87 2.03 1.85 1.86 1.88 Arginine 2.18 2.01 1.67 2.08 2.07 Aspartic Acid 3.44 3.29 2.82 3.30 3.35 Cysteine 0.48 0.45 0.39 0.45 0.47 Glutamic Acid 6.33 5.91 4.71 6.06 6.06 Glycine 1.56 1.56 1.42 1.46 1.39 Histidine 0.86 0.78 0.62 0.79 0.78 Hydroxylysine 0.08 0.07 0.08 0.05 0.05 Hydroxyproline 0.20 0.09 0.10 0.06 0.03 Isoleucine 1.60 1.53 1.27 1.51 1.51 Leucine 3.28 3.24 2.67 3.16 3.17 Lysine 2.01 2.02 1.78 2.01 2.00 Methionine 0.64 0.73 0.67 0.66 0.69

229

Phenylalanine 1.85 1.79 1.51 1.76 1.79 Proline 2.13 1.97 1.62 1.93 1.92 Serine 1.48 1.51 1.30 1.52 1.56 Threonine 1.29 1.31 1.16 1.28 1.29 Tryptophan 0.47 0.40 0.38 0.43 0.44 Tyrosine 1.33 1.25 1.02 1.27 1.25 Valine 1.73 1.81 1.54 1.74 1.72 1 Proximate composition and mineral composition was tested at Midwest Laboratories (Omaha, NE, USA). 2 Mineral composition was tested at Midwest Laboratories (Omaha, NE, USA).

23

0

Tab

le 7

c Pe

rfor

man

ce o

f ju

veni

le P

acifi

c w

hite

shr

imp

L. v

anna

mei

(In

itial

wei

ght 0

.15g

) of

fere

d di

ets

form

ulat

ed to

eva

luat

e U

lva

mea

l 4 a

s a

repl

acem

ent f

or s

oybe

an m

eal a

nd fi

sh m

eal o

n an

iso-

nitro

gen

basi

s in

juve

nile

shr

imp

over

six

wee

ks (T

rial 4

).

Die

t U

lva

Leve

ls (%

) Fi

nal b

iom

ass

(g)

Fina

l mea

n w

eigh

t (g)

W

G3 (%

) FC

R2

Surv

ival

(%)

PRE4 (%

)

T 4D

1 0

42.6

8a 4.

74a

3160

.39a

1.72

c 90

.0ab

30

.50a

T 4D

2 12

27

.85bc

3.

41bc

20

57.9

1bc

2.52

b 82

.5ab

20

.68bc

T 4D

3 24

20

.08d

2.72

c 17

18.6

7c 3.

26a

74.0

ab

15.9

1c

T 4D

4 4.

75

34.6

0b 3.

69b

2335

.98b

2.23

bc

94.0

a 25

.11ab

T 4D

5 9.

5 24

.84cd

3.

52bc

22

54.0

4bc

2.51

b 72

.0b

22.3

1b

PSE1

0.90

41

0.11

35

76.7

9 0.

0826

2.

4965

<0

.000

1

P-va

lue

<0.0

001

<0.0

001

<0.0

001

<0.0

001

0.01

07

0.78

82

1 Poo

led

stan

dard

err

or.

2 FC

R: F

eed

conv

ersi

on ra

tio =

Fee

d of

fere

d / (

Fina

l wei

ght -

Initi

al w

eigh

t).

3 WG

: Wei

ght g

ain

= (F

inal

wei

ght -

Initi

al w

eigh

t) / I

nitia

l wei

ght ×

100

%.

4 PR

E: P

rote

in re

tent

ion

effic

ienc

y =

(Fin

al w

eigh

t × F

inal

pro

tein

con

tent

) - (I

nitia

l wei

ght ×

Initi

al p

rote

in c

onte

nt) ×

100

/ Pr

otei

n of

fere

d.

Val

ues

with

in a

col

umn

with

diff

eren

t sup

ersc

ripts

are

sig

nific

antly

diff

eren

t bas

ed o

n Tu

key’

s m

ultip

le ra

nge

test

.

231

Table 7d Proximate2 composition of shrimp at the conclusion of a 6-week growth trial in which shrimp were offered diets formulated to evaluate Ulva meal 4 as a replacement for soybean meal and fish meal on an iso-nitrogen basis in juvenile shrimp (Trial 3).

Diet Moisture (%) Crude protein (%) Crude lipid (%) Crude fiber (%) Ash (%)

T4D1 76.1b 70.83b 8.40a 5.25 11.50c

T4D2 77.9a 73.02ab 5.07bc 4.98 12.69bc

T4D3 78.2a 73.76a 3.65c 5.57 14.26a

T4D4 76.1b 70.95b 6.90ab 5.34 12.11bc

T4D5 76.9ab 71.73ab 6.17b 5.45 12.94ab

P-value 0.0006 0.0027 <0.0001 0.2712 0.0001

PSE1 0.1937 0.3056 0.2541 0.0976 0.1669 1 Pooled standard error. 2 Body samples were analyzed at University of Missouri-Columbia, Agriculture Experiment Station Chemical Laboratory (Columbia, MO, USA). Values within a column with different superscripts are significantly different based on Tukey’s multiple range test.

232

Table 8a Composition of reference diet for the determination of digestibility coefficients of Ulva meal 1 and 2.

Ingredients % as is

Soybean meal1 10.00

Fish meal2 32.50

Fish oil2 3.20

Whole wheat3 47.60

Trace mineral premix4 0.50

Vitamin premix5 1.80

Choline cloride6 0.20

Stay C7 0.10

Corn starch3 1.00

Lecethin8 1.00

Chromic oxide9 1.00 1 De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA. 2 Omega Protein Inc., Houston TX, USA. 3 MP Biomedicals Inc., Solon, OH, USA 4 Trace mineral premix(g/100g premix): Cobalt chloride, 0.004; Cupric sulfate pentahydrate, 0.550; Ferrous sulfate, 2.000; Magnesium sulfate anhydrous, 13.862; Manganese sulfate monohydrate, 0.650; Potassium iodide, 0.067; Sodium selenite, 0.010; Zinc sulfate heptahydrate, 13.193; Alpha-cellulose, 69.664. 5 Vitamin premix (g/kg premix): Thiamin.HCL, 4.95; Riboflavin, 3.83; Pyridoxine.HCL, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; folic acid, 4.00; Cyanocobalamin, 0.05; Inositol, 25.00; Vitamin A acetate (500,000 IU/g), 0.32; Vitamin D3 (1,000,000 IU/g), 80.00; Menadione, 0.50; Alpha-cellulose, 856.81. 6 VWR, Radnor, PA, USA. 7 Stay C®, (L-ascorbyl-2-polyphosphate 35% Active C), DSM Nutritional Products., Parsippany, NJ, USA. 8 The Solae Company, St. Louis, MO, USA. 9 Alfa Aesar, Haverhill, MA, USA

23

3

Tab

le 8

b A

ppar

ent d

ry m

atte

r (A

DM

), ap

pare

nt e

nerg

y (A

ED)

and

appa

rent

pro

tein

(A

PD)

dige

stib

ility

val

ues

for

the

diet

(D

) an

d in

gred

ient

(I) u

sing

70:

30 re

plac

emen

t tec

hniq

ue o

ffer

ed to

Pac

ific

whi

te s

hrim

p (L

. van

nam

ei)

Mea

ns

AD

MD

A

EDD

A

PDD

A

DM

I A

EDI

APD

I

Bas

al d

iet 1

76

.38

± 0.

37a

82.6

5 ±

1.20

a 92

.08

± 0.

55a

So

ybea

n m

eal

77.0

2 ±

0.87

a 82

.63

± 1.

05ab

94

.76

± 0.

49a

78.5

1 ±

2.89

a 82

.56

± 3.

79a

97.0

3 ±

0.83

a

Fish

mea

l 1

68.2

1 ±

3.80

b 78

.31

± 3.

21bc

80

.86

± 1.

80b

49.1

5 ±

12.6

7b 69

.77

± 9.

51a

67.0

7 ±

4.02

b

Ulv

a m

eal 1

62

.19

± 1.

26c

71.9

6 ±

0.89

d 75

.14

± 1.

19d

29.1

0 ±

4.19

c 40

.39

± 3.

52b

15.1

7 ±

5.41

d

Bas

al d

iet 2

75

.69

±0.5

2a 81

.51

± 0.

41ab

92

.04

± 0.

03a

Fish

mea

l 2

67.9

9 ±

0.17

b 76

.44

± 0.

78c

82.3

4 ±

0.31

b 49

.45

± 0.

56b

65.7

8 ±

2.23

a 71

.30

± 0.

68b

Ulv

a m

eal 2

64

.63

± 1.

08bc

69

.99

± 0.

64d

78.3

3 ±0

.42c

38.2

6 ±

3.61

bc

19.1

1 ±

3.33

c 43

.51

± 1.

49c

Ulv

a m

eal 1

and

Ulv

a m

eal 2

repr

esen

t firs

t and

sec

ond

batc

h U

lva

mea

l. Fi

sh m

eal 1

and

Fis

h m

eal 2

repr

esen

t the

firs

t and

sec

ond

colle

ctio

n of

fish

mea

l die

t, re

spec

tivel

y.

Bas

al d

iet 1

and

Bas

al d

iet 2

repr

esen

t the

firs

t and

sec

ond

colle

ctio

n of

bas

al d

iet,

resp

ectiv

ely.

V

alue

s ar

e pr

esen

ted

as m

ean

± st

anda

rd d

evia

tion.

V

alue

s w

ithin

a c

olum

n w

ith d

iffer

ent s

uper

scrip

ts a

re s

igni

fican

tly d

iffer

ent o

n Tu

key’

s m

ultip

le ra

nge

test

.

233

Table 8c Apparent amino acids (AA) digestibility value for the soybean meal (SBM), fish meal (FM), Ulva meal 1 (UM1) and Ulva meal 2 (UM2) using 70:30 replacement technique offered to Pacific white shrimp (L. vannamei)

AA SBM FM UM1 UM2

Alanine 93.75 ± 2.02a 69.09 ± 4.09b 36.90 ± 6.56d 50.77 ± 1.10c

Arginine 96.91 ± 1.44a 75.35 ± 3.78b 42.20 ± 6.60c 47.21 ± 0.77c

Aspartic Acid 95.39 ± 1.36a 69.23 ± 3.70b 35.87 ± 5.69c 38.09 ± 1.70c

Cysteine 91.29 ± 1.68a 54.39 ± 7.06b 13.44 ± 10.85c 6.66 ± 7.45c

Glutamic Acid 95.69 ± 1.52a 70.84 ± 3.70b 33.85 ± 7.24c 23.25 ± 3.17c

Glycine 95.06 ± 2.05a 66.55 ± 6.26b 29.84 ± 8.78c 34.04 ± 4.96c

Histidine 94.33 ± 1.69a 74.26 ± 2.86b 7.10 ± 1.87d 43.52 ± 0.22c

Isoleucine 93.23 ± 1.72a 68.72 ± 3.99b 39.15 ± 5.74c 46.33 ± 0.79c

Leucine 92.23 ± 1.96a 71.29 ± 3.16b 34.65 ± 8.50d 50.43 ± 0.80c

Lysine 95.03 ± 1.84a 76.97 ± 2.24b 40.65 ± 6.50c 38.07 ± 3.04c

Methionine 95.20 ± 1.54a 70.63 ± 3.30b 44.13 ± 5.12c 40.89 ± 3.18c

Phenylalanine 93.41 ± 1.90a 65.28 ± 4.13b 27.23 ± 7.02d 47.25 ± 0.76c

Proline 94.68 ± 1.92a 67.21 ± 5.39b 15.81 ± 10.45c 18.20 ± 2.42c

Serine 93.11 ± 1.91a 58.31 ± 4.65b 10.76 ± 11.00c 43.41 ± 0.82b

Threonine 91.99 ± 1.94a 66.33 ± 3.35b 32.83 ± 6.84c 42.57 ± 0.26c

Tryptophan 95.37 ± 1.92a 80.31 ± 1.53b 65.58 ± 2.46c 70.84 ± 3.26c

Tyrosine 95.28 ± 1.22a 73.62 ± 3.40b 36.51 ± 4.10d 59.02 ± 0.45c

Valine 90.78 ± 2.39a 67.06 ± 3.75b 29.94 ± 6.89d 54.20 ± 0.42c

Total AA 94.31 ± 1.67a 69.91 ± 3.89b 29.80 ± 6.68d 41.67 ± 0.51c

Values are presented as mean ± standard deviation. Values within a row with different superscripts are significantly different on Tukey’s multiple range test.

234

Figure 1 (a) In trial 1, relationship between weight gain (y) of shrimp and incorporation levels of

Ulva meal 1 levels (x) in the diets. The regression line is described by y = 0.1686x3 - 6.2114x2 +

34.409x + 1797.8 (R2 = 0.4922, P < 0.0001). (b) In trial 1, relationship between FCR (y) and

supplemental Ulva meal levels (x) in the diets. The regression line is described by y = 0.0276x +

1.7708 (R2 = 0.3582, P = 0.0001). (c) In trial 1, relationship between lipid content (y) of shrimp

body and supplemental Ulva meal levels (x) in the diets. The regression line is described by y = -

0.0962x + 7.4 (R2 = 0.3503, P = 0.0002).

y = 0.1686x3 - 6.2114x2 + 34.409x + 1797.8R² = 0.4922

0

500

1000

1500

2000

2500

0 5 10 15 20 25 30

Perc

entw

eigh

tgai

n(%

)

Ulva meal levels (%)

(a)

y = 0.0276x + 1.7708R² = 0.35823

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25 30

FCR

Ulva meal levels (%)

(b)

y = -0.0962x + 7.4R² = 0.3503

0

2

4

6

8

10

12

0 5 10 15 20 25 30

Lipi

dco

nten

t(%

)

Ulva meal levels (%)

(c)

235

Figure 2 (a) In trial 2, relationship between weight gain (y) and Ulva meal 2 levels (x) in the

diets. The regression line is described by y = 1.1925x2 -62.699x + 1713.1 (R2 = 0.6815, P <

0.0001). (b) In trial 2, relationship between FCR (y) and supplemental Ulva meal 2 levels (x) in

the diets. The regression line is described by y = -0.0703x + 1.5451 (R2 = 0.4766, P < 0.0001). (c)

In trial 2, relationship between survival (y) and supplemental Ulva meal 2 levels (x) in the diets.

The regression line is described by y = -1.1607x + 100.27 (R2 = 0.6113, P < 0.0001). (d) In trial

2, relationship between lipid content (y) of shrimp body and supplemental Ulva meal 2 levels (x)

in the diets. The regression line is described by y = 0.0078x2 - 0.4095x + 7.8033 (R2 = 0.9051, P

< 0.0001).

y = 1.1925x2 - 62.699x + 1713.1R² = 0.68146

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40

Wei

ghtg

ain

(%)

Ulva meal levels (%)

(a) y = 0.0703x + 1.5451R² = 0.4766

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 10 20 30 40

FCR

Ulva meal levels (%)

(b)

y = -1.1607x + 100.27R² = 0.6113

0

20

40

60

80

100

120

0 10 20 30 40

Surv

ival

(%)

Ulva meal levels (%)

(c)

y = 0.0078x2 - 0.4095x + 7.8033R² = 0.9051

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 10 20 30 40

Lipi

dco

nten

t(%

)

Ulva meal levels (%)

(d)

236

Figure 3 (a) In trial 4, relationship between weight gain (y) of shrimp and incorporation levels of

Ulva meal 4 levels (x) in the diets. The regression line is described by y = 2.6848x2 - 119.17x +

3049.3 (R2 = 0.6934, P < 0.0001). (b) In trial 4, relationship between FCR (y) and supplemental

Ulva meal 4 levels (x) in the diets. The regression line is described by y = 0.06x + 1.8532 (R2 =

0.721, P < 0.0001). (c) In trial 4, relationship between lipid content (y) of shrimp body and

supplemental Ulva meal 4 levels (x) in the diets. The regression line is described by y = -0.1903x

+ 7.9555 (R2 = 0.7224, P < 0.0001).

y = 2.6848x2 - 119.17x + 3049.3R² = 0.6934

0

500

1000

1500

2000

2500

3000

3500

4000

0 5 10 15 20 25 30

Wei

ghtg

ain

(%)

Ulva meal levels (%)

(a)

y = 0.06x + 1.8532R² = 0.721

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25 30

FCR

Ulva meal levels (%)

(b)

y = -0.1903x + 7.9555R² = 0.7224

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 5 10 15 20 25 30

(c)